TW202011982A - Peptide formulations and uses thereof - Google Patents

Abstract

The present disclosure relates to compositions comprising a peptide with improved solubility and the methods of achieving the same. In one embodiment, the present composition comprises at least one peptide, a pH modifier present at a concentration of less than 1.0 mM, and a pharmaceutically acceptable carrier. The composition may further comprise other components such as immunomodulator adjuvant. Methods of preparing the present compositions and methods of treating a subject by administering the present compositions were also disclosed.

Description

Peptide formulations and uses

There is increasing interest in developing cancer therapies that contain neoplastic vaccines that are tumor-specific and/or patient-specific neoantigens. These new antigens are ideal for enhancing the response of T cells to specific tumor types, thus overcoming some of the disadvantages of vaccines containing shared tumor antigens. However, most neoantigenic peptides are difficult to dissolve in most pharmaceutically acceptable carriers, which hinders their use in vaccines or other pharmaceutical formulations. Poor solubility can cause poor absorption after administration, limit the shelf life of the formulation and reduce its efficacy after recovery. The present invention is directed to the development of formulations for improving the solubility of neoantigen peptides and to achieve the use of previously insoluble neoantigen peptides for pharmaceutical formulations.

Provided herein is a pharmaceutical composition comprising: one or more neoantigenic peptides or a pharmaceutically acceptable salt thereof; a pH adjusting agent present at a concentration of less than 1.0 mM; and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition is a vaccine composition.

In some embodiments, the pharmaceutical composition is aqueous.

In some embodiments, the pharmaceutical composition is lyophilizable.

In some embodiments, at least one of the one or more neoantigenic peptides is defined by pI> 5 and HYDRO> -6. As used herein, "by definition" is equivalent to "having". For example, a peptide defined by pI> 5 and HYDRO <-6 is equivalent to a peptide having pI> 5 and HYDRO <-6. In some embodiments, at least one of the one or more neoantigenic peptides is defined by pI>8 and HYDRO>-8. In some embodiments, at least one of the one or more neoantigenic peptides is defined by pI <5 and HYDRO> -5. In some embodiments, at least one of the one or more neoantigenic peptides is defined by pI> 9 and HYDRO <-8. In some embodiments, at least one of the one or more neoantigenic peptides is defined by pI> 7 and the value of HYDRO> -5.5. In some embodiments, at least one of the one or more neoantigenic peptides is defined by pI> 0 and HYDRO <-8, pI> 0 and HYDRO> -4, or pI> 4.3 and -4 ≤ HYDRO ≥ -8. In some embodiments, at least one of the one or more neoantigenic peptides is defined by pI> 0 and HYDRO> -4, or pI> 4.3 and HYDRO ≤ -4. In some embodiments, at least one of the one or more neoantigenic peptides is defined by pI> 0 and HYDRO> -4, or pI> 4.3 and -4 ≤ HYDRO ≥ -9. In some embodiments, at least one of the one or more neoantigenic peptides is defined by 5 ≤ pI ≥ 12 and -4 ≤ HYDRO ≥ -9. In some embodiments, at least one of the one or more neoantigenic peptides is defined by pI <4.3 and -4 ≤ HYDRO ≥ -8.

In some embodiments, at least one of the one or more neoantigenic peptides of the pharmaceutical composition has 5 ≤ pI ≥ 12 and -6 ≤ HYDRO ≥ -9.

In some embodiments, at least one of the one or more neoantigenic peptides of the pharmaceutical composition has 7.5 ≤ pI ≥ 12 and -4 ≤ HYDRO ≥ -9.

In some embodiments, at least one of the one or more neoantigenic peptides of the pharmaceutical composition has a pi of 5 to 6.5 or 7.5 to 12 and -4 ≤ HYDRO ≥ -9.

In some embodiments, at least one of the one or more neoantigenic peptides of the pharmaceutical composition has 5 ≤ pI ≥ 12 and has a HYDRO of -4 to -4.9 or -5.8 to -9.

In some embodiments, at least one of the one or more neoantigenic peptides of the pharmaceutical composition has a pi of 5 to 6.5 or 7.5 to 12 and a HYDRO of -4 to -4.9 or -5.8 to -9.

In some embodiments, the pharmaceutical composition comprises at least one neoantigenic peptide. In some embodiments, the pharmaceutical composition comprises at least two neoantigenic peptides. In some embodiments, the pharmaceutical composition comprises at least three neoantigenic peptides. In some embodiments, the pharmaceutical composition comprises at least four neoantigenic peptides. In some embodiments, the pharmaceutical composition comprises at least five neoantigenic peptides. In some embodiments, the pharmaceutical composition contains up to 40 neoantigenic peptides. In some embodiments, each of the one or more neoantigenic peptides is soluble in the pharmaceutical composition.

In some embodiments, each of the one or more neoantigenic peptides includes 6 to 50 amino acids, 6 to 45 amino acids, 6 to 40 amino acids, 6 to 35 amino acids, 6 To 30 amino acids, 6 to 25 amino acids, 6 to 20 amino acids, 8 to 50 amino acids, 8 to 45 amino acids, 8 to 40 amino acids, 8 to 35 Amino acids, 8 to 30 amino acids, 8 to 25 amino acids, 8 to 20 amino acids, 14 to 50 amino acids, 14 to 45 amino acids, 14 to 40 amines Base acids, 14 to 35 amino acids, 14 to 30 amino acids, 14 to 27 amino acids, 14 to 25 amino acids, or 14 to 20 amino acids. In some embodiments, at least one of the one or more neoantigenic peptides contains 14 to 30 amino acids. In some embodiments, at least one of the one or more neoantigenic peptides is less than 15 amino acids in length or less, 7 to 11 amino acids in length, or 8 to 10 amino acids in length.

In some embodiments, at least one of the one or more neoantigen peptides is 40 amino acids or less in length, 6 to 25 amino acids in length, 14 to 30 amino acids in length, or is 14 to 27 amino acids.

In some embodiments, the pH adjusting agent is a base. In some embodiments, the pH adjusting agent is a weak acid conjugate base. In some embodiments, the pH adjusting agent is a pharmaceutically acceptable salt. In some embodiments, the pH adjuster is a dicarboxylate or tricarboxylate. In some embodiments, the pH adjusting agent is citric acid and/or citrate. In some embodiments, the citrate salt is disodium citrate and/or trisodium citrate. In some embodiments, the pH adjusting agent is succinic acid and/or succinate. In some embodiments, the succinate salt is disodium succinate and/or monosodium succinate. In some embodiments, the disodium succinate is disodium succinate hexahydrate.

In some embodiments, the pH adjusting agent is present at a concentration of 0.75 mM or lower. In some embodiments, the pH adjusting agent is present at a concentration of 0.50 mM or less. In some embodiments, the pH adjusting agent is present at a concentration of 0.25 mM or lower. In some embodiments, the pH adjusting agent is present at a concentration of 0.25 mM to 1.0 mM. In some embodiments, the pH adjusting agent is present at a concentration of 0.50 mM to 1.0 mM. In some embodiments, the pH adjusting agent is present at a concentration of 0.75 mM to 1.0 mM. In some embodiments, the pH adjusting agent is present at a concentration of 0.50 mM to 0.75 mM.

In some embodiments, the pharmaceutically acceptable carrier contains a liquid. In some embodiments, the pharmaceutically acceptable carrier comprises water. In some embodiments, the pharmaceutically acceptable carrier comprises sugar. In some embodiments, the sugar comprises dextrose. In some embodiments, dextrose is present at a concentration of 5% w/v. In some embodiments, the sugar comprises trehalose. In some embodiments, the sugar comprises sucrose. In some embodiments, the pharmaceutically acceptable carrier comprises dimethyl sulfoxide (DMSO). In some embodiments, DMSO is present at a concentration of 0.1% to 10%, 0.5% to 5%, or 1% to 3%.

In some embodiments, the pharmaceutical composition further comprises an immunomodulator or adjuvant. In some embodiments, the immunomodulator or adjuvant is selected from the group consisting of poly ICLC, 1018 ISS, aluminum salt, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, ARNAX, STING agonist, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, carrier system, PLGA particles, resiquimod, SRL172, virus particles and others Virus-like particles, YF-17D, VEGF capture agent, R848, β-glucan, Pam2Cys, Pam3Cys and Aquila QS21 stimulator. In some embodiments, the immunomodulator or adjuvant comprises poly-ICLC. In some embodiments, the ratio of poly-ICLC to neoantigen peptide in the pharmaceutical composition is from 2:1 to 1:10 v:v. In some embodiments, the ratio of poly-ICLC to neoantigen peptide in the pharmaceutical composition is about 1:1, 1:2, 1:3, 1:4, or 1:5 v:v.

Provided herein is a pharmaceutical composition comprising: one or more neoantigen peptides; dimethyl sulfoxide present at a concentration of at least 0.5%, 1%, 2%, 3% or more; at least 1% w/v, 2 % w/v, 3% w/v, 4% w/v, 5% w/v or more sugars; and succinic acid or succinate in concentrations less than 1.0 mM.

Provided herein is a pharmaceutical composition comprising: one or more neoantigenic peptides; 1% to 10% dimethylsulfoxide; 1% w/v to 10% w/v sugar in water; and less than 1.0 mM succinic acid or Succinate.

Provided herein is a pharmaceutical composition comprising: one or more neoantigenic peptides; 1% to 10% dimethylsulfoxide; 1% w/v to 10% w/v sugar in water; and less than 1.0 mM succinic acid or Succinate; and Poly I: Poly C. In some embodiments, poly I:poly C comprises poly-L-lysine; in some embodiments, poly I:poly C comprises sodium carboxymethyl cellulose. In some embodiments, the pharmaceutical composition further comprises sodium chloride.

Provided herein is a pharmaceutical composition comprising: one to five neoantigenic peptides, 1% to 4% v/v dimethylsulfoxide, 3% to 5% dextrose, succinic acid present at a concentration of less than 1.0 mM or Succinate.

Provided herein is a pharmaceutical composition comprising: one to five neoantigenic peptides, 1% to 4% v/v dimethylsulfoxide, 3% to 5% dextrose, succinic acid present at a concentration of less than 1.0 mM or Succinate, 0.4 to 0.6 mg/ml poly I: Poly C, 0.3 to 0.5 mg/ml poly-L-lysine, 0.5 to 2 mg/ml sodium carboxymethyl cellulose and 0.1% to 0.5% chlorine Sodium chloride.

In some embodiments, each of the one or more neoantigenic peptides is present at a concentration of at least 1 μg/mL, at least 10 μg/mL, at least 25 μg/mL, at least 50 μg/mL, or at least 100 μg/mL. In some embodiments, each of the one or more neoantigenic peptides is at most 5000 μg/mL, at most 2500 μg/mL, at most 1000 μg/mL, at most 750 μg/mL, at most 500 μg/mL, at most 400 μg /mL or at most 300 μg/mL. In some embodiments, each of the one or more neoantigenic peptides ranges from 10 μg/mL to 5000 μg/mL, 10 μg/mL to 4000 μg/mL, 10 μg/mL to 3000 μg/mL, 10 μg/mL Concentrations of mL to 2000 μg/mL, 10 μg/mL to 1000 μg/mL, 25 μg/mL to 500 μg/mL, or 50 μg/mL to 300 μg/mL are present. In some embodiments, a higher percentage of neoantigen peptides are soluble in the pharmaceutical composition than the pharmaceutical composition containing a pH adjusting agent at a concentration of 1.0 mM or higher. In some embodiments, a higher percentage of one or more neoantigen peptides is soluble in the pharmaceutical composition than the pharmaceutical composition containing the pH adjusting agent present at a concentration of 1.0 mM or higher.

Provided herein is a vaccine comprising the pharmaceutical composition provided herein. In some embodiments, the vaccine is a neoplastic vaccine.

Provided herein are immunogenic compositions comprising the pharmaceutical compositions provided herein.

Provided herein is a vaccination or immunization kit comprising: a lyophilized composition containing one or more neoantigenic peptides; a pH adjuster; and instructions for combining (a) and (b) into a solution, where the solution contains the concentration PH adjuster less than 1.0 mM. In some embodiments, the kit further comprises a viral vector. In some embodiments, the kit further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is water.

Provided herein is a vaccination or immunization kit comprising: a composition comprising one or more lyophilized neoantigenic peptides or a pharmaceutically acceptable salt thereof; a pH adjusting agent; and combining (a) and (b) to a solution Instructions, where the solution contains a pH adjuster with a concentration of less than 1.0 mM.

In some embodiments, the vaccination or immunization kit further comprises an adjuvant. In some embodiments, the vaccination or immunization kit further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is water.

Provided herein is a method of preparing a pharmaceutical composition, which comprises combining one or more lyophilized neoantigen peptides with a pH adjusting agent and a pharmaceutically acceptable carrier to form a peptide solution, wherein the pH adjusting agent is present at a concentration of less than 1.0 mM . In some embodiments, the method further comprises filtering the peptide solution.

Provided herein is a method of preparing a pharmaceutical composition provided herein, which comprises combining one or more new antigen peptides with a pH adjusting agent and a pharmaceutically acceptable carrier to form a peptide solution, wherein the pH adjusting agent is less than 1.0 mM Concentration exists. In some embodiments, the method further comprises filtering the peptide solution. In some embodiments, one or more neoantigenic peptides are lyophilized before pooling.

Provided herein is a method of preparing a vaccine, which comprises combining the pharmaceutical composition provided herein with an immunomodulator or adjuvant.

Provided herein is a method of treating a subject suffering from a condition or disease, which comprises administering to the subject the pharmaceutical composition provided herein to treat the condition or disease.

In some embodiments, the method further comprises administering another therapeutic agent to the subject. In some embodiments, the method further comprises administering a second pharmaceutical composition to the subject, wherein the second pharmaceutical composition is the pharmaceutical composition provided herein. In some embodiments, the method further comprises administering a third pharmaceutical composition to the subject, wherein the third pharmaceutical composition is the pharmaceutical composition provided herein. In some embodiments, the method further comprises administering a fourth pharmaceutical composition to the subject, wherein the fourth pharmaceutical composition is the pharmaceutical composition provided herein. In some embodiments, the method further comprises administering at least a fifth, sixth, seventh, or eighth pharmaceutical composition to the subject, wherein the fifth, sixth, seventh, or eighth pharmaceutical composition is herein The provided pharmaceutical composition.

Provided herein are formulations of the pharmaceutical compositions provided herein for use in the preparation of medicaments for the treatment of conditions or diseases. In some embodiments, the condition or disease is a neoplasm. In some embodiments, the neoplasm is cancer.

Provided herein is an aqueous solution of neoantigen peptides, comprising: one or more neoantigen peptides; and the presence of succinic acid or succinate at a concentration of less than 1.0 mM.

Provided herein is a method for preparing a new antigen peptide aqueous solution, which comprises combining one or more new antigen peptides with succinic acid or succinate and a liquid carrier, wherein the succinic acid or succinate is present at a concentration of less than 1.0 mM .

Provided herein is a method for increasing the solubility of one or more neoantigen peptides, which includes adjusting the concentration of a pH adjusting agent present in one or more neoantigen peptides, a pH adjusting agent, and a pharmaceutically acceptable carrier In the pharmaceutical composition; wherein the adjustment includes reducing the concentration of the pH adjusting agent to a concentration of less than 1.0 mM.

Cross reference

This application claims the priority of US Provisional Application No. 62/660,027 filed on April 19, 2018; it is incorporated herein by reference in its entirety. References incorporated

Reference is made to International Patent Application No. PCT/US2014/068893 filed on December 5, 2014, which claims priority to US Provisional Patent Application No. 61/913,172 filed on December 6, 2013.

With reference to the serial number of the international patent application PCT/US2016/036605 filed on June 9, 2016, it claims priority to the US Provisional Patent Application No. 62/172,890 filed on June 9, 2015.

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated for reference The way into the general.

Some specific details of this specification are set forth in order to provide a thorough understanding of various embodiments. However, those skilled in the art will understand that the present invention can be practiced without such details. In other cases, well-known structures have not been shown or described in detail to avoid unnecessarily obscure the description of the embodiments.

Unless the context requires otherwise, throughout this specification and subsequent patent applications, the word "comprise" and its variations (such as "comprises and comprising") should be regarded as open and inclusive. It is regarded as "including but not limited to". In addition, the headings provided in this article are for convenience only and do not explain the scope or meaning of the claimed disclosure.

Unless the context clearly indicates otherwise, the singular forms "a/an" and "the" include plural references as used in this specification and the appended patent applications. It should also be noted that, unless the context clearly indicates otherwise, the term "or" is generally adopted in the sense that it includes "and/or".

The term "about" or "approximately" means within an acceptable error range for a particular value as determined by those of ordinary skill in the art, which will depend in part on how the value is measured or determined, This is the limitation of the measurement system. For example, according to the practice in this technology, "about" may mean within 1 or greater than 1 standard deviation. Alternatively, "about" may mean a range of at most 20%, at most 10%, at most 5%, or at most 1% of the predetermined value. Or, especially in terms of biological systems or methods, the term may mean within an order of magnitude of a certain value, preferably within 5 times and more preferably within 2 times. When a specific value is described in the scope of this application and the patent application, unless otherwise stated, it should be assumed that the term "about" means within an acceptable error range of the specific value. Unless the context clearly dictates otherwise, all numerical values provided herein are modified by the term about.

The ranges provided herein are intended to include all values within that range. For example, the range 1 to 50 should be understood to include any number, combination of numbers, or sub-ranges from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and all intermediate decimal values between the aforementioned integers, such as (for example) 1.1, 1.2, 1.3, 1.4 , 1.5, 1.6, 1.7, 1.8 and 1.9. As far as sub-ranges are concerned, they specifically cover "nested sub-ranges" that extend from either end of the range. For example, the nested sub-ranges of the exemplary range 1 to 50 may include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, in the other direction, 50 to 20 and 50 to 10.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of various aspects described herein, suitable methods and materials are described below. definition

The term "analog" is considered to mean molecules that are different but have similar functional or structural characteristics. For example, a tumor-specific neoantigen polypeptide analog maintains the biological activity of the corresponding naturally-occurring tumor-specific neoantigen polypeptide, while having certain biochemical modifications that can enhance the function of the analog relative to the naturally occurring polypeptide. These biochemical modifications can increase the protease resistance, membrane permeability or half-life of the analog without changing, for example, ligand binding. Analogs may include non-standard or non-natural amino acids.

The terms "neo-antigen" and "neoantigen" are used interchangeably. The terms "neo-antigenic" and "neoantigenic" are used interchangeably. The terms ``neo-antigen'', ``neoantigen'', ``neo-antigenic'' or ``neoantigenic'' may mean not encoded in the normal, non-mutated host genome Of antigen. Neoantigens can include one or more amino acid-modified antigens compared to the parent antigen. For example, the neoantigen may be a neoplasm-associated neoantigen, wherein the term "tumor-associated neoantigen" may include a peptide or protein including amino acid modifications caused by tumor-specific mutations. In some cases, neoantigens represent oncogenic viral proteins or abnormal proteins that are produced by somatic mutations. For example, new antigens can be produced by disrupting cellular mechanisms through the activity of viral proteins. Another example can be exposure to carcinogenic compounds, which in some cases can cause somatic mutations. This somatic mutation can eventually lead to the formation of a tumor/cancer.

The term "neoplastic tumor" is considered to mean any disease caused or caused by inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancer is an example of neoplasia. Examples of cancer include, but are not limited to: leukemia (eg, acute leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, acute myelogenous leukemia, acute promyeloid leukemia, acute myelomonocytic leukemia, acute monocytic leukemia Nuclear leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia); polycythemia vera; lymphoma (eg, Hodgkin's disease, non-Hodgkin's disease) ); Waldenstrom's macroglobulinemia; heavy chain diseases and solid tumors, such as sarcomas and carcinomas (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma) , Angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, triple negative breast cancer (TNBC), ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, marrow Carcinoma, bronchial carcinoma, renal cell carcinoma, liver tumor, nile duct carcinoma, choriocarcinoma, sperm cell carcinoma, embryonal carcinoma, Wilm's tumor, cervix Cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, neuroblastoma, craniopharyngioma, ependymoma, pine (Fruitoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannomas, meningiomas, melanoma, neuroblastoma and retinoblastoma). Lymphoproliferative disorders are also considered proliferative diseases.

The term "vaccine" may refer to a pharmaceutical preparation (pharmaceutical composition) or product that induces an immune response (especially a cellular immune response) that recognizes and attacks pathogens or diseased cells such as cancer cells immediately after administration. Vaccines can be used to prevent or treat diseases.

The term "neoplastic tumor vaccine" is intended to refer to a neoplastic/tumor-specific neoantigen combination, such as at least two, at least three, at least four, at least five or more neoantigenic peptides. "Vaccine" is understood to mean a composition used to generate immunity for the prevention and/or treatment of diseases (eg neoplasms/tumors). Therefore, a vaccine is an agent that contains an antigen and is intended for use by humans or animals to produce specific defense and protective substances through vaccination. The "tumor vaccine composition" may include pharmaceutically acceptable excipients, carriers or diluents.

The term "pharmacologically acceptable" may refer to approval or approval by a regulatory agency of the federal or state government, or it is listed in the United States Pharmacopoeia or other recognized pharmacopeia for animals (including humans).

"Pharmaceutically acceptable carrier, excipient or diluent" may refer to a carrier, excipient or diluent that can be administered to the subject together with the active ingredient, such as small molecule compounds, antibodies, nucleic acid molecules , Peptides, polypeptides and fragments thereof, and they do not destroy the pharmacological activity of the active ingredient.

The "pharmaceutically acceptable salt" of neoantigen peptides or combinations of neoantigen peptides may refer to the technology generally considered suitable for use in contact with human or animal tissues without excessive toxicity, irritation, allergic reactions or other problems Or complication of acid or alkali salts. Such salts include inorganic and organic acid salts of basic residues (such as amines), and basic or organic salts of acidic residues (such as carboxylic acids). Specific pharmaceutical salts include, but are not limited to, the following acid salts: such as hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, p-aminobenzenesulfonic acid, formic acid , Toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid , Salicylic acid, glutamic acid, ascorbic acid, pamoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid; alkane Acids such as acetic acid, HOOC-(CH ₂ ) _n -COOH, where n is 0 to 4, and similar acids. Similarly, pharmaceutically acceptable cations include but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium. The "pharmaceutically acceptable salts" of neoantigen peptides may also refer to those listed by Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, PA, page 1418 (1985). In general, pharmaceutically acceptable acid or base salts can be synthesized from the parent compound containing a basic or acidic moiety by any conventional chemical method. Briefly, these salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of a suitable base or acid in a suitable solvent.

The term "polypeptide" or "peptide" may refer to a polypeptide that has been separated from components that naturally accompany it. Unless otherwise specified, the term "polypeptide" or "peptide" covers lyophilized peptides and pharmaceutically acceptable salts of the peptides. Generally, the peptides provided herein have a purity of at least 60% by weight, free of proteins and naturally occurring organic molecules that are naturally associated with them. Alternatively, the peptide has a purity of at least 75% by weight, at least 90% by weight, or at least 99% by weight. Peptides can be obtained, for example, by extraction from natural sources, by expression of recombinant nucleic acids encoding such peptides; or by chemical synthesis. The purity of the peptide can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, and HPLC analysis.

The term "subject" may refer to an animal that is the target of treatment, observation or experiment. By way of example only, subjects include, but are not limited to: mammals, including but not limited to human or non-human mammals, such as non-human primates, bovines, equines, canines, ovine Or cats.

As used herein, the terms "prevent/preventing/prevention", "prophylactic treatment" and similar terms refer to those who are not suffering from, but at risk of suffering from a disease or condition or are susceptible to a disease or The probability of having a disease or condition in a subject with a condition is reduced.

The term "treat/treated/treating/treatment" and similar terms are intended to mean reducing or ameliorating the condition and/or symptoms associated with it (eg, neoplasms or tumors). "Treatment" includes the concept of "remission", which means reducing the frequency or severity of the occurrence or recurrence of any symptoms related to cancer or the effects of other diseases and/or side effects associated with cancer therapy. The term "treatment" also covers the concept of "management", which refers to reducing the severity of a patient's specific disease or condition or delaying its recurrence, for example, prolonging the remission time of patients already suffering from the disease. It should be understood that although not excluded, the treatment of a condition or condition does not require the complete elimination of the condition, condition or symptoms related thereto.

"Decrease" means a negative change of at least 10%, 25%, 50%, 75% or 100%.

The term "curative effect" refers to a certain degree of alleviation of a condition (eg, neoplasm or tumor) or one or more symptoms of its associated pathology. As used herein, "therapeutically effective amount" refers to an amount of an agent that, after single or multiple dose administration to cells or subjects, effectively prolongs the survival period of patients with such disorders and reduces one of the disorders Or more symptoms and symptoms, prevention or delay (and similar effects) than expected in the absence of such treatment. "Therapeutically effective amount" is intended to limit the amount required to achieve a therapeutic effect. A physician or veterinarian with general skills can easily determine and specify the "therapeutically effective amount" of the required pharmaceutical composition (eg, ED50). For example, a physician or veterinarian may start a dosage of the compound used in the pharmaceutical composition below the level required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

Pharmaceutical compositions should generally provide a dosage of about 0.0001 mg to about 200 mg of compound per kg of body weight per day. For example, systemic doses to human patients can range from 0.01 to 10 µg/kg, 20 to 80 mg/kg, 5 to 50 µg/kg, 75 to 150 µg/kg, 100 to 500 mg/kg, 250 to 750 µg/kg, 500 to 1000 µg/kg, 1 to 10 mg/kg, 5 to 50 mg/kg, 25 to 75 mg/kg, 50 to 100 mg/kg, 1.00 to 250 mg/kg, 50 To 100 mg/kg, 250 to 500 mg/kg, 500 to 750 mg/kg, 750 to 1000 mg/kg, 1000 to 1500 mg/kg, 1500 to 2000 mg/kg, 5 mg/kg, 20 mg/kg , 50 mg/kg, 1.00 mg/kg or 200 mg/kg. Pharmaceutical unit dosage forms are prepared to provide about 0.001 to about 5000 mg (eg, about 100 to about 2500 mg) compound or combination of basic ingredients per unit dosage form. Neoplasm / tumor specific antigen

The present invention relates to vaccines and the treatment of neoplasms by administering a therapeutically effective amount of a pharmaceutical composition (e.g., cancer vaccine) comprising a plurality of neoplasms/tumor-specific neoantigens to a subject (e.g., mammals, such as humans) (and More specifically the tumor) method. As described herein, whole-genome/exome sequencing can be used to identify all or nearly all mutated neoantigens that are particularly present in neoplasms/tumors of individual patients, and the set of mutated neoantigens can be analyzed to identify the neoantigens Specific, optimized subset for use as a personalized cancer vaccine or immunogenic composition for treating neoplasms/tumors of patients. For example, by sequencing neoplastic/tumor and normal DNA of each patient, a population of neoplastic/tumor-specific neoantigens can be identified to identify tumor-specific mutations, and the patient’s human leukocyte antigen (HLA) isoform can be identified . A validated algorithm can then be used to perform bioinformatics analysis on neoplastic/tumor-specific neoantigen populations and their cognate primary antigens to predict which tumor-specific mutations form an epitope that can bind to the patient’s HLA heterotype . Based on this analysis, multiple peptides corresponding to a subset of these mutations can be designed and synthesized for each patient and combined together to be used as a cancer vaccine or immunogenic composition to immunize the patient. The neoantigenic peptide can be combined with an adjuvant (eg poly-ICLC) or another anti-neoplastic agent. Without being bound by theory, these new antigens are expected to bypass central thymus tolerance (thus allowing a stronger anti-tumor T cell response) while reducing the possibility of autoimmunity (eg, by avoiding targeting normal self-antigens).

The immune system can be classified into two functional subsystems: innate and acquired immune systems. The innate immune system is the first line of defense against infection, and most probable pathogens are quickly neutralized by the system before it can cause, for example, a significant infection. The acquired immune system reacts with the molecular structure (called antigen) of the invading organism. There are two types of innate immune responses, humoral immune responses and cell-mediated immune responses. In the humoral immune response, antibodies secreted by B cells into the humor bind to pathogen-derived antigens, allowing pathogens to be eliminated via various mechanisms, such as complement-mediated lysis. In a cell-mediated immune response, T cells that can destroy other cells are activated. For example, if a disease-associated protein is present in a cell, it is proteolytically split into peptides within the cell. The specific cell protein then attaches itself to the antigen or peptide formed in this way and transfers it to the surface of the cell. Once there is a molecular defense mechanism that presents it to the body, especially T cells. Cytotoxic T cells recognize these antigens and kill the cells that carry the antigen.

Molecules that transfer and display peptides on the cell surface are called major histocompatibility complex (MHC) proteins. The MHC protein is divided into two types: called MHC class I and MHC class II. Although the structures of the two MHC classes of proteins are very similar, they have very different functions. Class I MHC proteins are present on the surface of almost all cells (including most tumor cells) of the upper body. Class I MHC proteins are loaded with antigens usually derived from endogenous proteins or from pathogens present in the cells, and then presented to native or cytotoxic T-lymphocytes (CTL). Class II MHC proteins are present on dendritic cells, B lymphocytes, macrophages and other antigen presenting cells. It mainly presents peptides processed from external antigen sources (ie, from outside the cell to T-helper (Th) cells). Most of the peptides bound by class I MHC proteins are derived from cytoplasmic proteins produced in the organism's own healthy host cells and generally do not stimulate immune responses. Therefore, cytotoxic T-lymphocytes recognizing that these self-peptides exhibit MHC class I molecules are missing in the thymus (central tolerance), or are missing or inactivated after their release from the thymus (i.e., tolerance (peripheral resistance Acceptability)). MHC molecules can stimulate immune responses when they present peptides to non-tolerant T lymphocytes. Cytotoxic T lymphocytes have T cell receptor (TCR) and CD8 molecules on their surface. T cell receptors can recognize and bind peptides complexed with class I MHC molecules. Each cytotoxic T lymphocyte exhibits a unique T cell receptor capable of binding a specific MHC/peptide complex.

Peptide antigens attach themselves to MHC class I molecules by competitive affinity binding within the endoplasmic reticulum before they appear on the cell surface. Here, the affinity of individual peptide antigens is directly related to the amino acid sequence and the presence of specific binding motifs in defined positions within the amino acid sequence. Therefore, if the sequence of such peptides is known, it may be possible to use, for example, peptide vaccines to manipulate the immune system against diseased cells.

One of the key obstacles to the development of curative and tumor-specific immunotherapy is the identification and selection of highly specific and restricted tumor antigens to avoid autoimmunity. Tumor neoantigens generated as a result of genetic changes (such as reversals, translocations, deletions, missense mutations, splice site mutations, etc.) within malignant cells represent most tumor-specific classes of antigens. At present, due to technical difficulties in identifying new antigens, selecting and optimizing new antigens, and producing new antigens for use in vaccines or immunogenic compositions, new antigens have rarely been used in cancer vaccines or immunogenic compositions. These problems can be overcome by using whole genomes, whole exomes (e.g. only captured exons), or tumors to identify tumors at the DNA level by comparing RNA sequencing of matched germline samples from each patient/ All or nearly all mutations in the tumor; use one or more peptide-MHC binding prediction algorithms to analyze the identified mutations in order to generate a plurality of candidate neoantigen T cell antigens that appear in neoplasms/tumor and can bind to the patient's HLA allele Determinant; and synthesis of a plurality of candidate neoantigen peptides selected from all neoORF peptides and predicted binding peptide groups for use in cancer vaccines or immunogenic compositions.

For example, translating sequencing information into therapeutic vaccines can include: (1) Personal mutant peptides predicted to bind to HLA molecules of individuals. Effective selection of which specific mutation to use as an immunogen requires identifying the patient's HLA type and being able to predict which mutant peptide will effectively bind to the patient's HLA allele. Recently, learning methods based on neural networks and verified binding peptides and non-binding peptides have improved the accuracy of prediction algorithms for major HLA-A and HLA-B alleles. (2) The drug is formulated as a long peptide multi-epitope vaccine. In fact, targeting as many mutant epitopes as possible takes advantage of the enormous power of the immune system to prevent immune escape opportunities by down-regulating specific immune targeting gene products, and to compensate for known inaccuracies in the way epitopes are predicted. Synthetic peptides provide a particularly suitable way to efficiently prepare multiple immunogens and can quickly translate the identified mutant epitopes into an effective vaccine. By using reagents or animal substances that do not contain contaminating bacteria, peptides can be easily synthesized chemically and peptides can be easily purified. The small size causes a definite lesion on the mutant domain of the protein and also reduces unrelated antigen competition from other components (such as non-mutant proteins or viral vector antigens).

Merged with strong vaccine adjuvant. Effective vaccines may require strong adjuvants to elicit an immune response. As described herein, poly-ICLC (an agonist of TLR3 and the RNA helicase domain of MDA5 and RIG3) has been demonstrated to have several desirable properties required for vaccine adjuvants. These properties include, but are not limited to: in vivo induction of local and systemic activation of immune cells, production of chemoattractants and cytokines, and stimulation of antigen presentation by DCs. In addition, poly-ICLC can induce long-lasting CD4+ and CD8+ responses in humans. Notably, surprising similarities in the upregulation of transcription and signal transduction pathways were observed in subjects who had been vaccinated with poly-ICLC and in volunteers who had received highly effective, replication-competent yellow fever vaccines. In addition, in the latest Phase 1 study, >90% of ovarian cancer patients immunized with the combination of poly-ICLC and NY-ESO-1 peptide vaccine (except Montanide) showed the induction of CD4+ and CD8+ T cells and the antibody-to-peptide reaction. Ultimately, polyICLC has been fully tested in over 25 clinical trials to date and exhibits a relatively benign toxicity profile. Further advantages are described in this article.

As described herein, there is significant evidence in animals and humans that mutant epitopes are effective in inducing immune responses and that spontaneous tumor regression or long-term survival is related to CD8+ T cell responses to mutant epitopes (Buckwalter and Srivastava PK . "It is the antigen(s), stupid" and other lessons from over a decade of vaccitherapy of human cancer. Seminars in immunology 20:296-300 (2008); Karanikas, etc., High frequency of cytolytic T lymphocytes directed against a tumor -specific mutated antigen detectable with HLA tetramers in the blood of a lung carcinoma patient with long survival. Cancer Res. 61:3718-3724 (2001); Lennerz et al., The response of autologous T cells to a human melanoma is dominated by mutated neoantigens . Proc Natl Acad Sci USA.102:16013 (2005)), and "immune editing" can track changes in the expression of dominant mutant antigens in mice and humans (Matsushita et al., Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting Nature 482:400 (2012); DuPage et al., Expression of tumor-specific antigens underlies cancer immunoediting Nature 482:405 (2012); and Sampson et al., Immunologic escape after prolonged progression-free survival with epidermal growth factor receptor variant III peptide vaccination in patients with newly diagnosed glioblastoma J Clin Oncol. 28:4722-4729 (2010)). In some embodiments, mutant epitopes of cancer patients are determined.

In some embodiments, the mutant epitope is determined by sequencing the genome and/or exome of tumor tissue and healthy tissue from cancer patients using next-generation sequencing technology. In some embodiments, next-generation sequencing technology is used to sequence genes, which are selected based on their mutation frequency and ability to act as a new antigen. Next-generation sequencing can be applied to genome sequencing, genome re-sequencing, transcriptome analysis (RNA sequencing), DNA-protein interaction (ChIP sequencing), and epigenome characterization (de Magalhaes JP, Finch CE, Janssens G (2010). -generation sequencing in aging research: emerging applications, problems, pitfalls and possible solutions". Ageing Research Reviews 9 (3): 315-323; Hall N (May 2007). "Advanced sequencing technologies and their wider impact in microbiology". J . Exp. Biol. 209 (Pt 9): 1518-1525; Church GM (January 2006). "Genomes for all". Sci. Am. 294 (1): 46-54; ten Bosch JR, Grody WW (2008) . "Keeping Up with the Next Generation". The Journal of Molecular Diagnostics 10 (6): 484-492; Tucker T, Marra M, Friedman JM (2009). "Massively Parallel Sequencing: The Next Big Thing in Genetic Medicine". The American Journal of Human Genetics 85 (2): 142-154). Next-generation sequencing can now quickly reveal the presence of discrete mutations (such as coding mutations) in individual tumors, the most common monoamino acid changes (such as missense mutations), and insertion/deletion/gene fusion, stop codes by frame shift Unusual stretches of neo-amino acids resulting from translational mutations in the son and translation of improperly spliced introns (eg, neoORF). NeoORF is particularly valuable as an immunogen because its entire sequence is completely new to the immune system and thus resembles viral or bacterial foreign antigens. Therefore, neoORF: (1) is highly specific for tumors (that is, there is no expression in any normal cells); and (2) can bypass central tolerance, thereby increasing the frequency of neoantigen-specific CTL precursors . For example, recently the use of peptides derived from human papilloma virus (HPV) has demonstrated the possibility of using similar foreign sequences in therapeutic anti-cancer vaccines or immunogenic compositions. Approximately 50% of 19 patients with pre-neoplastic, virus-induced diseases who received 3 to 4 mixed vaccines of HPV peptides derived from viral oncogenes E6 and E7 maintained a complete response> 24 months (Kenter et al. , Vaccination against HPV-16 Oncoproteins for Vulvar Intraepithelial Neoplasia NEJM 361:1838 (2009)).

Sequencing technology has revealed that tumors contain multiple, patient-specific mutations that alter the protein coding content of genes. These mutations produce altered proteins ranging from monoamino acid changes (due to missense mutations) to new amino groups caused by reading frame shifts, through reading of blocked codons or translation of intron domains Addition of the long domain of the acid sequence (new open reading frame mutation; neoORF). These mutant proteins are valuable targets for the host's immune response to the tumor. This is because unlike the native protein, it has not been immune-suppressed by its own tolerance. Therefore, the mutant protein is more likely to be immunogenic and more specific to tumor cells than normal cells of the patient.

An alternative method for identifying tumor-specific neoantigens is through direct protein sequencing. Enzymatically decomposed protein sequencing using multidimensional MS technology (MSn) including tandem mass spectrometry (MS/MS) can also be used to identify new antigens. These proteomic approaches permit rapid and highly automated analysis (see, for example, K. Gevaert and J. Vandekerckhove, Electrophoresis 21:1145-1154 (2000)). Further encompassing high-throughput methods for de novo sequencing of unknown proteins can be used to analyze the proteome of a patient's tumor in order to identify expressed new antigens. For example, inter shotgun protein sequencing can be used to identify expressed new antigens (see, for example, Guthals et al. (2012) Shotgun Protein Sequencing with Meta-contig Assembly, Molecular and Cellular Proteomics 11(10):1084-96).

MHC multimers can also be used to recognize neoantigen-specific T cell responses to recognize neoplasm-specific neoantigens. For example, high-throughput analysis of neoantigen-specific T cell responses in patient samples can use MHC tetramer-based screening techniques (see, for example, Hombrink et al. (2011) High-Throughput Identification of mutant epitopes to an effective vaccine or immunogenic composition). By using reagents or animal substances that do not contain contaminating bacteria, peptides can be easily chemically synthesized and peptides can be easily purified. The small size causes a clear focus on the mutant domain of the protein and also reduces unrelated antigen competition from other components (unmutated protein or viral vector antigen).

In some embodiments, the drug formulation is a long peptide multiple epitope vaccine or immunogenic composition. These "long" peptides undergo effective internalization, are processed and cross-presented in professional antigen presenting cells (such as dendritic cells), and have been shown to induce CTL in humans (Melief and van der Burg, Immunotherapy of established (pre) malignant disease by synthetic long peptide vaccines Nature Rev Cancer 8:351 (2008)). In some embodiments, at least one neoantigenic peptide is prepared for immunization. In some embodiments, at least 2 new antigen peptides are prepared for immunization. In some embodiments, at least 3 new antigen peptides are prepared for immunization. In some embodiments, at least 4 new antigen peptides are prepared for immunization. In some embodiments, at least 5 new antigen peptides are prepared for immunization. In some embodiments, 20 or more peptides are prepared for immunization. In some embodiments, up to 40 new antigen peptides are prepared for immunization. In some embodiments, the neoantigen peptide has a length of about 5 to about 50 amino acids, a length of about 5 to about 45 amino acids, a length of about 5 to about 40 amino acids, and a length of about 5 Up to about 35 amino acids, about 5 to about 30 amino acids in length, about 5 to about 25 amino acids in length, about 5 to about 20 amino acids in length, about 10 to about 50 amino acids, about 10 to about 45 amino acids in length, about 10 to about 40 amino acids in length, about 10 to about 35 amino acids in length, about 10 to about 30 in length Amino acids, about 10 to about 25 amino acids in length, about 10 to about 20 amino acids in length, about 15 to about 50 amino acids in length, and about 15 to about 45 amino groups in length Acid, about 15 to about 40 amino acids in length, about 15 to about 35 amino acids in length, about 15 to about 30 amino acids in length, about 15 to about 25 amino acids in length, About 15 to about 20 amino acids in length, about 14 to about 50 amino acids in length, about 14 to about 45 amino acids in length, about 14 to about 40 amino acids in length, and From about 14 to about 35 amino acids, from about 14 to about 30 amino acids in length, from about 14 to about 25 amino acids in length, or from about 14 to about 20 amino acids in length. In some embodiments, peptides with a length of about 14 to about 35 amino acids are synthesized. In some embodiments, peptides with a length of about 14 to about 27 amino acids are synthesized. In some embodiments, the length of the neoantigenic peptide is in the range of about 20 to about 35 amino acids. The generation of tumor-specific neoantigens

The invention is based at least in part on the ability to present a patient's immune system with a combination of tumor-specific neoantigens. Those skilled in the art, based on the present invention and the knowledge in this technology, should understand that there are various methods for generating these tumor-specific new antigens. In general, these tumor-specific neoantigens can be produced in vitro or in vivo. Tumor-specific neoantigens can be produced in vitro as peptides or polypeptides, which can then be formulated into personalized neoplastic vaccines or immunogenic compositions and administered to subjects. These in vitro productions can be performed by various methods known to those skilled in the art, such as, for example, peptide synthesis or peptides from DNA or RNA molecules in any of a variety of bacterial, eukaryotic or viral recombinant expression systems /Peptide performance, then purify the expressed peptide or polypeptide. Alternatively, tumor-specific neoantigens can be generated in vivo by introducing molecules encoding tumor-specific neoantigens (eg, DNA, RNA, viral expression systems, and the like) into subjects, and then expressing the encoded tumor-specific neoantigens in vivo New antigen. Method for preparing formulations

The present invention relates to pharmaceutical compositions with improved solubility of neoantigen peptides. The solubility of a peptide describes the nature of the peptide in a specific solution and under specific conditions. Solubility can also refer to the maximum amount of peptide that can be dissolved in a certain amount of a particular solvent and under certain circumstances. The solubility of a peptide may depend on the structure, composition and other characteristics of the peptide, including but not limited to the number of residues in the peptide, the size and molecular weight of the peptide, the distribution and amount of hydrophilic and hydrophobic residues, the negative charge on the peptide and the positive The distribution and amount of charge and the isoelectric point (pI) of the peptide. For example, when all other properties are equal, peptides with shorter residues may have higher solubility than peptides with longer residues. The solubility of the peptide can also depend on the nature of the solvent, such as its pH, hydrophobicity, hydrophilicity, polar boiling point, etc. When all other properties are equal, peptides with continuous hydrophobic residues are more insoluble in hydrophilic solvents than peptides without continuous hydrophobic residues. Some hydrophobic and hydrophilic peptides that are insoluble in pure water may be soluble in polar organic solvents, such as dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) and acetonitrile; the relatively high polarity of their solvents And the dipole moment can make it more suitable for dissolving hydrophobic and/or charged peptides than pure water. In some cases, the solvent may be a mixture of different solvents (individual solvents in the mixture, "co-solvents"). Each co-solvent is soluble in the solvent mixture, and if the co-solvent is insoluble, stabilizers such as emulsifiers, surfactants, and viscosity modifiers may be present in the solvent to improve its homogeneity. The choice of solvent can be limited by the application of the peptide. For example, a mixture of an aqueous solution and a water-soluble organic solvent (eg, water/DMSO) can be used for injectable formulations. Restrictions on the use of organic solvents in injectable formulations may also include possible precipitation, pain, inflammation, and hemolysis after injection. In some cases, the choice of solvent can be further limited by the stability of the peptide in the solvent; the solvent should not react with the peptide or promote the degradation of the peptide. In addition, the solubility of a peptide in a particular solvent may depend on temperature, pressure, the method and steps taken to dissolve the peptide, and so on. In the typical steps of dissolving peptides, mild sonic treatment and heating can be adopted, and the solubility of peptides can vary depending on the order, addition rate, and how to combine the co-solvent with the peptide.

The solubility of the peptide can be determined experimentally. In some embodiments, visual inspection of a solution containing a peptide (a clear solution without precipitation) means that the peptide can be dissolved in that particular solvent or solvent mixture under a specified set of conditions (eg, fixed temperature and pressure). In some embodiments, the turbidity of the peptide-containing solution can be used to determine whether the peptide is soluble in a particular solvent or solvent mixture under a specified set of conditions. In some embodiments, turbidity may be defined as a measure of the clarity of a fluid, liquid, or solution, and is represented by the amount of light or radiation (eg, visible light, infrared radiation) scattered when passing through the fluid, liquid, or solution . In some embodiments, the turbidity of the peptide-containing solution can be qualitatively determined by visual inspection. In some embodiments, turbidity can be measured using any instrument or technique suitable for measuring turbidity. Such instruments and technologies include but are not limited to turbidity meters or turbidity sensors. In some embodiments, the turbidity of a solution containing peptides is approximately the same as its reference composition but does not contain peptides. In some embodiments, when the turbidity values of the peptide-containing solution and the reference solution are approximately equal, the peptide is considered to be soluble in the solution under a specific set of conditions. In some embodiments, when it is determined that the turbidity value of the peptide solution exceeds the turbidity value of the reference solution and when the difference of the determined turbidity values is statistically significant, the peptide is considered partially soluble or insoluble The solution.

其中N_i 為N末端及精胺酸(R)、離胺酸(K)及組胺酸(H)之側鏈的數量及pKa_i 為N末端及精胺酸(R)、離胺酸(K)及組胺酸(H)之側鏈的pKa值，且N_j 為C末端及天冬胺酸(D)、麩胺酸(E)、半胱胺酸(C)及酪胺酸(Y)胺基酸之側鏈的數量及pKa_j 為C末端及天冬胺酸(D)、麩胺酸(E)、半胱胺酸(C)及酪胺酸(Y)胺基酸之側鏈的pKa值。上文所列之帶電酸的側鏈及肽之胺基及羧基末端之pKa值為吾人所知(表1)。在上式中，對於既定肽(具有如上文所描述之pKa_i 及pKa_j 值)Z = 0時之pH值為彼既定肽之pI。表 1 ：胺基酸側鏈pKa值(表根據Lehninger Principles of Biochemistry, 2008, Macmillan重建)

The solubility of a peptide in an aqueous solution can also be predicted based on its amino acid sequence without experimentation. In some embodiments, the solubility of a peptide in an aqueous solution can be predicted based on the pi and hydrophobicity of the peptide. In some embodiments, the solubility of the peptide can be predicted by using commonly available software programs. Peptides with certain pI and hydrophobic properties are more likely to be soluble in aqueous solutions than peptides with other pI and hydrophobic properties. The isoelectric point (pI) of a peptide is the pH at which the net charge of the peptide is zero. Peptides possess amino acid residues that can be positive, negative, or neutral, and the sum of the charges on each alloamino acid or terminal is the net charge of the peptide. The net charge (Z) of a peptide at a certain pH can be estimated by the following formula:

Where N _i is the N-terminus and the number of side chains of arginine (R), lysine (K) and histidine (H) and pKa _i is the N-terminus and arginine (R), lysine ( K) and the pKa value of the side chain of histidine (H), and N _j is the C terminal and aspartic acid (D), glutamic acid (E), cysteine (C) and tyrosine ( Y) The number of amino acid side chains and pKa _j are C-terminal and aspartic acid (D), glutamic acid (E), cysteine (C) and tyrosine (Y) amino acids The pKa value of the side chain. The side chains of the charged acids listed above and the pKa values of the amine and carboxyl ends of the peptides are known to us (Table 1). In the above formula, the pH value for a given peptide (with pKa _i and pKa _j values as described above) Z=0 is the pI of the given peptide. Table 1 : pKa values of amino acid side chains (table reconstructed according to Lehninger Principles of Biochemistry, 2008, Macmillan)

The pI of a peptide can be estimated by using a calculator, which is easy to obtain online (eg, provided by pepcalc.com). At a pH below its pI, the peptide carries a net positive charge, and at a pH above its pI The peptide carries a net negative charge. Therefore, the pI of the peptide can affect its solubility. In some embodiments, the solubility of the peptide in aqueous solution can be minimized by adjusting the pH of the solution so that it corresponds to the pI of the peptide.

The hydrophobicity of the peptide can be determined by various methods known to those skilled in the art. In some embodiments, the hydrophobicity of the peptide may use the Kyte-Doolittle method (e.g., see Jack Kyte and Russell) for progressively evaluating the hydrophilicity and hydrophobicity of the peptide along the amino acid sequence of the peptide F. Doolittle, J. Mol. Biol. (1982) 157, 105-132). This method uses the moving fragment method to continuously measure the average hydrophilicity as it progresses through the sequence within fragments with a predetermined length. Continuous scores are drawn to the carboxyl terminus. The midpoint line corresponds to the total average value of the hydrophilicity of the peptide and in some embodiments is called the Kate-Dullit hydrophobicity value. Individual amine groups used in the Kate-Dullit method The known hydrophobicity fraction of the acid is provided in Table 2.

Table 2 : Amino Acid Hydrophobicity Fractions Used in Kate-Dulit Method

In some embodiments, the hydrophobicity of the peptide can be determined by calculating the hydrophobicity index of each hydrophobic domain of the peptide and then identifying the domain with the highest hydrophobicity. First, identify the different hydrophobic amino acid domains in the peptide that contain one or more sequences. Thereafter, the total hydrophobicity score or hydrophilicity score of each hydrophobic domain is calculated by adding together the hydrophobicity value/hydrophilicity value of each amino acid in the domain. This calculation can be performed using published values of the hydrophobicity or hydrophilicity of each amino acid side chain. For example, the hydrophilicity of each of the 20 standard amino acids is provided in Table 3. Positive values correspond to hydrophilic amino acids and negative values correspond to hydrophobic amino acids.

Table 3 : Hydrophilicity values

The domain of a hydrophobic amino acid can be defined as a domain in which all amino acids in the domain have a hydrophilicity value of less than zero. The sum of the hydrophilicity values of individual amino acids in a given hydrophobic sequence is the hydrophobicity index. The consecutive sequence with the largest negative hydrophobicity index is the most hydrophobic. In some embodiments, the hydrophobicity index is called HYDRO. In some embodiments, the hydrophobicity index of a peptide can be determined by identifying a domain that includes one or more consecutive amino acids (where each amino acid has a hydrophilicity value of less than 0), and where the The domain of multiple amino acids has a smaller (eg, more negative) total hydrophilicity value than any other domain of a peptide that contains one or more amino acids. For example, a peptide with the amino acid sequence LEYVAFSQRFIPEL (SEQ ID NO: 1) has a hydrophobicity index of -6.8, and a peptide with the amino acid sequence LARDIPPAVTGKWKLSDLRRYGAVPSG (SEQ ID NO: 2) has a hydrophobicity index of -3.4 .

Various approaches can be taken to increase the solubility of the peptide. In some embodiments, the solubility of the peptide can be increased by selecting suitable solvents (including solvent mixtures). Suitable solvents can be based on the physical properties of the peptide (including its isoelectric point (pI) and hydrophobicity, polarity) and/or identified by experiment. Suitable solvents can be pure solvents or solvent mixtures, and for the latter, the composition of the co-solvent can also be adjusted to increase the solubility of the peptide. In some embodiments, the co-solvent is miscible and forms a homogeneous solvent mixture. In some embodiments, one or more co-solvents may not be miscible with the solvent mixture and surfactants, emulsifiers, thickeners, or other stabilizers that may be required to extend the shelf life. In some embodiments, the solubility of the peptide can be increased by changing the pH of the solvent; for example, the pH of the solvent is adjusted away from the pI of the peptide by adding acid or base. In some embodiments, the solubility of the peptide can be increased by sonication, heating, and/or using a suitable dissolution procedure. In some embodiments, the solubility of the peptide in the DMSO/water mixture can be increased by first dissolving the peptide in DMSO and then adding water. In another embodiment, the solubility of the peptide may be via the addition of a complex agent such as cyclic oligosaccharide cyclodextrin or addition of such as n-butanol, ethanol, guanidine hydrochloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2 -The chaotropic agent of propanol, sodium lauryl sulfate, thiourea and urea increases. In some cases, the solubility of the peptide may depend on the ionic strength of the solvent. In some embodiments, the solubility can be increased by increasing the salt concentration in the solvent (salt solution). In some embodiments, the solubility may increase as the salt concentration in the solvent decreases (salting out).

In a simple composition such as a composition containing only one peptide and one solvent, the relationship between the solubility of the peptide and the change in the composition can be easily determined. However, in complex compositions such as pharmaceutical or vaccine compositions, it may be difficult to determine the effect of composition changes on peptide solubility. Changes in the pharmaceutical composition can simultaneously increase peptide solubility through one mechanism and reduce peptide solubility through another mechanism; for example, increasing the salt content in the composition can reduce peptide solubility by increasing ionic strength (salt Analysis), but at the same time can increase the solubility of the peptide due to changes in pH.

One aspect of the invention relates to a method for increasing the solubility of a peptide. In some embodiments, the invention relates to a method for increasing the solubility of neoantigen peptides. It was unexpectedly found that reducing the concentration of the pH adjusting agent in the compositions provided herein can increase the solubility of at least one neoantigenic peptide. In some embodiments, the method of increasing the solubility of the peptide includes adjusting or reducing the concentration of the pH adjusting agent to less than 1.0 mM. In some embodiments, the method of increasing the solubility of the peptide comprises adding a pH adjusting agent to the composition comprising at least one neoantigenic peptide and a pharmaceutically acceptable carrier so that the concentration of the pH adjusting agent is lower than 1.0, 0.95, 0.90, 0.85, 0.80, 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10 mM or less.

In some embodiments, the solubility of at least one neoantigenic peptide in the composition provided herein at a defined temperature and the composition of the at least one neoantigenic peptide in a composition in which the concentration of the pH adjusting agent is 1.0 mM or greater Increase in solubility by at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000%. In some embodiments, the solubility of at least one neoantigenic peptide in the compositions provided herein and the at least one neoantigenic peptide in a composition in which the concentration of the pH adjusting agent is 1.0 mM or greater at a defined temperature The solubility increases by approximately 10% to approximately 300%, approximately 20% to approximately 300%, approximately 50% to approximately 300%, approximately 100% to approximately 300%, approximately 150% to approximately 300%, approximately 200% to approximately 300 %, about 250% to about 300%, about 10% to about 250%, about 20% to about 250%, about 50% to about 250%, about 100% to about 250%, about 150% to about 250%, About 200% to about 250%, about 10% to about 200%, about 20% to about 200%, about 50% to about 200%, about 100% to about 200%, about 150% to about 200%, 10% To about 150%, 20% to about 150%, 50% to about 150%, 100% to about 150%, 10% to about 100%, 20% to about 100%, 50% to about 100%, 10% to About 50%, 20% to about 50%, 10% to about 20%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%. In some embodiments, the solubility of at least one neoantigenic peptide in the compositions provided herein and the at least one neoantigenic peptide in a composition in which the concentration of the pH adjusting agent is 1.0 mM or greater at a defined temperature The solubility is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 Times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times , About 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times. In some embodiments, the defined temperature is ambient temperature. In some embodiments, the defined temperature is about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, At about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 50°C, about 55°C, about 60°C, about 65°C, or about 70°C.

In some embodiments, at least one neoantigen peptide in the pharmaceutical composition provided herein is insoluble, partially soluble, or soluble in the composition at a given temperature.

In some embodiments, at least one neoantigen peptide in the composition provided herein is soluble in the composition at ambient temperature. In some embodiments, at about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C , About 15 ℃, about 20 ℃, about 25 ℃, about 30 ℃, about 35 ℃, about 40 ℃, about 50 ℃, about 55 ℃, about 60 ℃, about 65 ℃, about 70 ℃, about 75 ℃, about At 80°C, about 85°C, about 90°C, about 95°C, or about 100°C, at least one neoantigen peptide in the composition provided herein is soluble in the composition. In some embodiments, at about 35 °C, about 35.5 °C, about 36 °C, about 36.5 °C, about 36.7 °C, about 36.8 °C, about 36.9 °C, about 37 °C, about 37.1 °C, about 37.2 °C, about 37.3 °C , About 37.4 ℃, about 37.5 ℃, about 37.6 ℃, about 37.7 ℃, about 37.8 ℃, about 37.9 ℃, about 38 ℃, about 38.5 ℃, about 39 ℃, about 39.5 ℃ or about 40 ℃ combinations provided herein At least one of the new antigen peptides is soluble in the composition. Pharmaceutical composition

Provided herein is a pharmaceutical composition comprising at least one neoantigenic peptide. The present invention also relates to vaccine compositions and peptide solutions containing at least one neoantigen peptide. In some embodiments, the pharmaceutical composition of the present invention may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more new Antigen peptides.

Unless otherwise excluded, "peptide" or "neoantigen peptide" as used herein includes its pharmaceutically acceptable salts and lyophilized forms of the peptide. Neoantigen peptides can be identified and synthesized by any suitable method known in the art or described herein. In some embodiments, the at least one neoantigenic peptide contains about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 amino acid residues. In some embodiments, the at least one neoantigen peptide is from about 5 to about 50, from about 5 to about 45, from about 5 to about 40, from about 5 to about 35, from about 5 to about 30, from about 5 to about 25, about 10 to about 50, about 10 to about 45, about 10 to about 40, about 10 to about 35, about 10 to about 30, about 10 to about 25, about 15 to about 50, about 15 to about 45, about 15 to About 40, about 15 to about 35, about 15 to about 30, about 15 to about 25, about 20 to about 50, about 20 to about 45, about 20 to about 40, about 20 to about 35, about 20 to about 30 Or in the range of about 20 to about 25 amino acids. In some embodiments, the at least one neoantigen peptide is about 6 to about 25, about 15 to about 35, about 15 to about 24, about 9 to about 15, about 8 to about 11, or about 9 to about 10 amines in length Within the base acid range. In some embodiments, the length of at least one neoantigenic peptide is equal to or less than 30, 25, 20, or 15 amino acid residues. In some embodiments, the length of each neoantigen peptide is independent of the length of other neoantigen peptides (if present) that make up the pharmaceutical composition.

The ideal concentration of each peptide in the composition of the present invention can be determined by those skilled in the art based on the specific application of the pharmaceutical composition. For example, the concentration and amount of each peptide can vary depending on the condition of the subject, the method of administration, and/or the solubility of the peptide. In some embodiments, each of one to five neoantigen peptides or pharmaceutically acceptable salts thereof is at about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 μg/mL, Concentrations of 1000 μg/mL, 2000 μg/mL, 3000 μg/mL, 4000 μg/mL or above exist. In some embodiments, the total concentration of neoantigen peptides in the pharmaceutical composition is about 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500 μg/mL or above. In some embodiments, each of the one or more neoantigen peptides or pharmaceutically acceptable salts thereof ranges from 10 μg/mL to 4000 μg/mL, 10 μg/mL to 3000 μg/mL, 10 μg/mL To 2000 μg/mL, 10 μg/mL to 1000 μg/mL, 10 μg/mL to 500 μg/mL, 10 μg/mL to 300 μg/mL, 25 μg/mL to 500 μg/mL or 50 μg/mL A concentration of up to 300 μg/mL is present in the pharmaceutical composition.

The at least one neoantigen peptide may be soluble or may be insoluble in the pharmaceutical composition. In some embodiments, the solubility of each neoantigen peptide is independent of the solubility of other neoantigen peptides. In some embodiments, the solubility of each neoantigen peptide is related to the solubility of other neoantigen peptides. In some embodiments, each of the neoantigenic peptides can be soluble in pharmaceutical compositions. In some aspects, the present invention relates to increasing the solubility of at least one peptide; therefore, in some embodiments, it contains the same pH adjusting agent and the same pharmaceutically acceptable load present at a concentration of 1.0 mM or higher. The percentage of at least one neoantigen peptide in the pharmaceutical composition of the drug is soluble in the pharmaceutical composition compared to a higher percentage of at least one neoantigen peptide. In some embodiments, the percentage of at least one neoantigen peptide soluble in the pharmaceutical composition is about 5% to about 100%, about 10% to about 100%, about 15% to about 100%, about 20% to about 100%, about 25% to about 100%, about 30% to about 100%, about 35% to about 100%, about 40% to about 100%, about 45% to about 100%, about 50% to about 100% , About 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%. In some embodiments, all neoantigen peptides are insoluble. In some embodiments, at least one of the neoantigenic peptides is partially soluble in the pharmaceutical composition. In some embodiments, at least one of the neoantigenic peptides may be insoluble in the pharmaceutical composition. In some embodiments, at least one of the neoantigenic peptides can remain insoluble in the pharmaceutical composition in order to obtain controlled release or other application needs. In some embodiments, at least one of the neoantigen peptides can be stabilized in an emulsion or suspension.

Provided herein is a pharmaceutical composition comprising at least one soluble peptide and a method of selecting a soluble peptide. The solubility of peptides can be predicted based on their HYDRO and pI values. In some embodiments, at least one neoantigen peptide is defined by pI> 0 and HYDRO <-8, pI> 0 and HYDRO> -4 or pI> 4.3 and -4 and ≤ HYDRO ≥ -8. In some embodiments, at least one of the one or more neoantigenic peptides is defined by pI <4.3 and -4 ≤ HYDRO ≥ -8. In some embodiments, at least one neoantigen peptide is defined by pI> 0 and HYDRO> -4 or pI> 4.3 and HYDRO ≤ -4. In some embodiments, at least one neoantigen peptide is defined by pI> 0 and HYDRO> -4 or pI> 4.3 and -4 ≤ HYDRO ≥ -9. In some embodiments, at least one neoantigenic peptide is defined by 5 ≤ pI ≥ 12 and -4 ≤ HYDRO ≥ -9. In some embodiments, at least one neoantigenic peptide is defined by pI> 5 and HYDRO> -6. In some embodiments, at least one neoantigen peptide is defined by pI>8 and HYDRO>-8. In some embodiments, at least one neoantigenic peptide is defined by pI <5 and HYDRO> -5. In some embodiments, at least one neoantigen peptide is defined by pI> 9 and HYDRO <-8. In some embodiments, at least one neoantigenic peptide is defined by pI> 7 and HYDRO value> -5.5. Methods for selecting soluble peptides for use in pharmaceutical compositions can include determining the pI and HYDRO values of the peptide and comparing their pI and HYDRO values to the pI and HYDRO ranges provided herein.

Pharmaceutically acceptable salts may refer to appropriate salts or complexes of active compounds that retain the desired biological activity of the parent compound and exhibit limited toxicological effects on normal cells. A non-exhaustive list of examples of such salts includes (a) acid addition salts with inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and similar acids), and with organic acids (such as acetic acid , Oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid and polyglutamic acid, etc.); and (b) with metal cations (such as Alkali addition salts formed by zinc, calcium, sodium, potassium and similar metal cations, and many others). Each of the new antigen peptides can be independently modified to affect its efficacy, solubility, stability, bioavailability, metabolic rate, and/or other properties. Modification can be carried out chemically at the side chain or at the end or by binding the peptide to one or more carriers (eg proteins). Various types of neoantigen peptides may contain one or more epicenters and are therefore stereoisomers, racemates and racemic mixtures, single enantiomers, individual diastereomers and non-isomeric isomers Enantiomeric mixtures and/or cis-trans isomers appear. This document specifically includes all such isomeric forms of these compounds. The compounds of the present invention may also exist in multiple tautomeric, amorphous and/or crystalline forms, and all tautomeric, amorphous and crystalline forms of the compounds are expressly included in the present invention.

A pH adjusting agent refers to a compound or a combination of two or more compounds that can change the pH of the composition when added to the pharmaceutical composition. The pH adjusting agent may include pharmaceutically acceptable salts of acids, organic acids, inorganic acids, latent acids, weak acids, bases, conjugate bases, organic bases, inorganic bases, latent bases, acids or bases and/or combination. Potential acids or bases are compounds that can become acids or bases under certain conditions, such as after heating or in the presence of water. The pH adjusting agent may be in solid and/or liquid form and may be soluble, partially soluble or insoluble in the pharmaceutical composition. In some embodiments, the pH adjusting agent comprises an organic acid and/or a pharmaceutically acceptable salt thereof. Suitable organic acids may contain one or more acidic groups, such as carboxylic acid, sulfonic acid, phosphoric acid, phosphorous acid, and hypophosphorous acid groups. Non-exhaustive examples of suitable acids that can be included in the pH adjusting agent include mono-, di-, tri-, or poly-carboxylic acids, mono-, di-, tri-, or poly-sulfonic acids, sorbic acid, adipic acid, malonic acid , Succinic acid, glutaric acid, maleic acid, fumaric acid, gluconic acid, lactic acid, glycolic acid, ascorbic acid, malic acid, tartaric acid, hydroxymalonic acid, mucinic acid, citric acid, amino Acids, glutamic acid, aspartic acid, acids containing aromatic groups (such as benzoic acid, phthalic acid, isophthalic acid, salicylic acid, terephthalic acid and trimellitic acid), and polymerization Acids (such as poly(acrylic acid) and poly(methacrylic acid)). In some embodiments, the pH adjusting agent includes a base and/or a pharmaceutically acceptable salt thereof, such as a compound containing an amine group, a hydroxyl group, an amidino group, or a phosphazene group. In some embodiments, the pH adjuster comprises a dicarboxylate or tricarboxylate. In some embodiments, the pH adjusting agent is succinic acid, monosodium succinate, disodium succinate, or a combination thereof. In some embodiments, the disodium succinate is disodium succinate hexahydrate. In some embodiments, the pH adjusting agent is citric acid, disodium citrate, trisodium citrate, or a combination thereof. The cation constituting the salt of the pH adjuster may be independently selected for each salt, and the cation may be organic or inorganic. Non-exhaustive examples of cations include sodium, aluminum, calcium, lithium, magnesium, zinc, potassium, arginine, benzathine, chloroprocaine, choline, diethanolamine, ethanolamine, ethylenedioxide Amine, histidine, lysine, procaine and trimethylamine.

The concentration of the pH adjusting agent may be less than 1.0 mM. It has been unexpectedly found that a pH adjusting agent concentration below 1.0 mM provides increased solubility of the new antigen peptide in the pharmaceutical compositions provided herein compared to a pH adjusting agent concentration equal to or above 1.0 mM. In some embodiments, the pH adjuster is below or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, The concentration of 0.94, 0.95, 0.96, 0.97, 0.98 or 0.99 mM is present in the pharmaceutical composition. In some embodiments, the pH adjusting agent is below 1.0 mM and above about 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, A concentration of 0.90 or 0.95 mM is present in the pharmaceutical composition. In some embodiments, the pH adjuster is below about 0.75 mM and above about 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55 , 0.60, 0.65 or 0.70 mM concentrations are present in the pharmaceutical composition. In some embodiments, the pH adjusting agent is present in the pharmaceutical composition at a concentration below about 0.50 mM and above about 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, or 0.45 mM. It should be understood that depending on the nature of the pH adjusting agent (eg, pKa value), the appropriate concentration for one pH adjusting agent may not be suitable for different pH adjusting agents. When a person skilled in the art replaces the pH adjusting agent in the composition of the present invention, the required optimal concentration of the pH adjusting agent may vary accordingly.

An acceptable carrier as provided herein is physiologically acceptable for administration to a patient, and should retain the therapeutic properties of the active ingredient, the compound or drug administered with/in it. Acceptable carriers and their formulations are generally described in, for example, Remington' Pharmaceutical Sciences (18th Edition, Editor: A. Gennaro, Mack Publishing Co., Easton, PA 1990). A pharmaceutically acceptable carrier is a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transferring the active ingredient from The site of administration of one organ or body part to another organ or body part or in an in vitro assay system. The acceptable carrier is compatible with the other ingredients of the pharmaceutical composition and does not substantially change the specific activity of the neoantigen peptide. At the doses and concentrations used, acceptable carriers should be non-toxic and harmless to the subjects to whom they are administered. Depending on the application and the route of administration, acceptable carriers can be in gas, liquid or solid form. Acceptable carriers can refer to solvents, co-solvents, buffers, isotonic agents, binding agents or complexing agents, ionic and/or non-ionic surfactants, viscosity modifiers, including emulsions, suspensions, liposomes, polymerization Systems of micelles, microparticles, porous nanostructures, dendrimers, lipid-based nanoparticles, gels, cyclodextrins, carrier proteins, etc. or combinations thereof. The binding agent or complexing agent may be a compound that chemically or physically interacts with a component of the composition such that the stability, solubility, or other properties of the component change; for example, interacting and increasing at least one The compound of the solubility of the neoantigen peptide. Binders or complexing agents include, but are not limited to chelating agents, such as EDTA; proteins, such as keyhole serocyanin, bovine serum albumin, ovalbumin, gelatin, and immunoglobulins; cyclic oligosaccharides, such as cyclodextrin; and metals Complexes, such as Zn-protein complexes. Systems containing emulsions, suspensions, liposomes, polymer micelles, microparticles, porous nanostructures, dendrimers, lipid-based nanoparticles, gels, cyclodextrins, carrier proteins, etc. or combinations thereof are meant to include A composition of any of the above components or structures.

In some embodiments, the pharmaceutical composition includes a buffer. Buffer refers to a solution whose pH changes negligibly when a small amount of acidic or basic material is added thereto. Exemplary buffers include, but are not limited to phosphate, citrate, citric acid, acetic acid, borate, and histidine. In some embodiments, the composition of the present invention includes a surfactant. The surfactant may be nonionic, anionic, cationic, amphoteric, zwitterionic, or a combination thereof. Exemplary surfactants include, but are not limited to, fatty alcohol ethers and/or polyoxyalkylene alkyl ethers, polyoxyalkylene fatty acid esters, Tweens®, polysorbates, Pluronics®, and poloxamer, poly Alkylene oxide.

In some embodiments, the pharmaceutically acceptable carrier comprises water and/or at least one non-aqueous liquid. Examples of acceptable non-aqueous liquids include, but are not limited to DMSO, DMF, acetonitrile, alcohols (such as ethanol), and combinations thereof. In some embodiments, pharmaceutically acceptable carriers include sugars, including but not limited to: monosaccharides such as dextrose, glucose, fructose, and galactose; and disaccharides such as sucrose, lactulose, lactose , Maltose, trehalose, cellobiose and chitosan. Pharmaceutically acceptable carriers can also include polysaccharides such as starch, cellulose and chitin; and sugar alcohols such as mannitol and sorbitol. In some embodiments, the pharmaceutically acceptable carrier includes water and dextrose. The amount and concentration of pharmaceutically acceptable carriers in the pharmaceutical composition may depend on the nature of the other components here; for example, higher concentrations of DMSO may be suitable for pharmaceutical combinations that include more hydrophobic neoantigen peptides Thing. In some embodiments, the concentration of DMSO present in the pharmaceutical composition is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% by volume (% v/v) , 9%, 10% or more. In some embodiments, the concentration of sugar present in the pharmaceutical composition is about 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0% by weight (% w/w water) in water , 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5% or more. In some embodiments, the concentration of sugar present in the composition is about 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5 by weight of the solution (% w/w) %, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5% or more. In some embodiments, the concentration of sugar present in the composition is about 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0 by weight of water in the solution (% w/w water) %, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5% or more.

The pharmaceutical composition of the present invention may further contain other additives, such as diluents, fillers, binders, disintegrants, lubricants, colorants, preservatives, stabilizers, and agents that contribute to delayed release. Depends on the route of administration. The compositions provided herein can be administered orally, topically, parenterally (such as by intravenous), intramuscularly, subcutaneously, intraarterially, intraarticularly, intrathecally and intradermally, or by other applicable The way of giving. In some embodiments, the compositions of the present invention can be administered by injection of unit doses.

The pharmaceutical composition of the present invention can be a vaccine composition in which at least one neoantigen peptide can be used as a vaccine. In some embodiments, the vaccine composition may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more neoantigenic peptides. In some embodiments, the composition includes an immunomodulator or adjuvant. In some embodiments, the compositions of the present invention include two or more immunomodulators or adjuvants. An immunomodulator can refer to an agent that induces, amplifies, weakens, prevents, or otherwise changes the immune response or function of the immune system. An adjuvant may refer to an agent that modifies the immune function of another agent. The immunomodulators or adjuvants provided herein may include poly ICLC, polyinosinic acid: polycytidylic acid (poly I: C, or poly I: poly C), 1018 ISS, aluminum salt, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, ARNAX, STING agonist, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, single Phosphatidyl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, carrier system, PLGA particles, Resimod, SRL172, virus particles and other virus-like particles, YF-17D, VEGF capture agent, R848, β-glucan, Pam3CysAquila's QS21 stimulator, ketone derivatives (such as Vadimezan) Or AsA404) or a combination thereof. The adjuvant may also include acrylic (co)polymers, methacrylic (co)polymers, and/or copolymers of maleic anhydride and alkenyl derivatives. For example, such (co)polymers may be acrylic or methacrylic acid polymers crosslinked with polyalkenyl ethers of sugars or polyols (carbomers), allyl sucrose crosslinked Or a copolymer of acrylic acid or methacrylic acid with allyl pentaerythritol, a copolymer of maleic anhydride and ethylene crosslinked with divinyl ether. Alternatively, the immunomodulator or adjuvant may include cytokines (such as TNF-α, GM-CSF, IL-1, IL-4 and IL-12); Duo-like receptors (such as TLR4 and TLR9); chemically modified Qualitative CpGs (eg CpR, Idera); non-CpU bacterial DNA or RNA and immunologically active small molecules and antibodies, such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, Pazopanib (pazopanib), ZD2171, AZD2171, ipilimumab (ipilimumab), tremelimumab (tremelimumab) and SC58175. In some embodiments, the immunomodulator or adjuvant comprises poly-ICLC (poly-ICLC). The immunomodulator or adjuvant can be administered separately to the vaccine or pharmaceutical composition; alternatively, it can be administered separately but in parallel with the composition of the invention.

In some embodiments, the pharmaceutical composition includes: (a) one to five neoantigenic peptides or pharmaceutically acceptable salts thereof; (b) DMSO; (c) sugars such as dextrose; (d) less than 1.0 mM succinic acid or succinate; (E) poly I: poly C; (f) poly-L-ionic acid; (g) carboxymethyl cellulose; (h) chloride; and (i) water. In some embodiments, each of one to five peptides or a pharmaceutically acceptable salt thereof is present at a concentration of about 50, 100, 150, 200, 250, 300, 350, 400 μg/ml or more. In some embodiments, the pharmaceutical composition comprises (% v/v) equal to or lower than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of DMSO. In some embodiments, DMSO is present in the pharmaceutical composition at about 1% to 3% by volume (% v/v). In some embodiments, the pharmaceutical composition comprises a concentration of about 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8% by weight of the solution (% w/w) , 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5 %, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8% , 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5 %, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10% or more Spin sugar. In some embodiments, the pharmaceutical composition comprises a concentration of about 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7% by weight of water in the solution (% w/w water) , 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4 %, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7% , 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4 %, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10% or Dextrose above. In some embodiments, the pharmaceutical composition comprises about 3.6% to 3.7% dextrose by weight (% w/w) of the solution. In some embodiments, the pharmaceutical composition comprises about 3.6% to 3.7% dextrose by weight of water in the solution (% w/w water). In some embodiments, the pharmaceutical composition comprises Poly I: Poly C at a concentration of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg/ml or more. In some embodiments, the pharmaceutical composition comprises a concentration of about 0.100, 0.125, 0.150, 0.175, 0.200, 0.225, 0.250, 0.275, 0.300, 0.325, 0.350, 0.375, 0.400, 0.425, 0.450, 0.475, 0.500 mg/ml or The above poly-L-lysine. In some embodiments, the pharmaceutical composition comprises sodium carboxymethyl cellulose at a concentration of about 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50 mg/ml or more. In some embodiments, the pharmaceutical composition comprises a concentration of 0.100%, 0.125%, 0.150%, 0.175%, 0.200%, 0.225%, 0.250%, 0.275%, by weight of water in the solution (% w/w water) Sodium chloride of 0.300%, 0.325%, 0.350%, 0.375%, 0.400%, 0.425%, 0.450%, 0.475%, 0.500% or more. In some embodiments, the pharmaceutical composition comprises a concentration of 0.100%, 0.125%, 0.150%, 0.175%, 0.200%, 0.225%, 0.250%, 0.275%, 0.300%, by weight of the solution (% w/w) Sodium chloride of 0.325%, 0.350%, 0.375%, 0.400%, 0.425%, 0.450%, 0.475%, 0.500% or more.

In some embodiments, the pharmaceutical composition may be lyophilizable or lyophilized. In some embodiments, the lyophilized composition may have a longer shelf life and may be easier to transfer. In some embodiments, the shelf life of the lyophilized composition is about 0.5 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, About 30 minutes, about 45 minutes, or about 60 minutes. In some embodiments, the shelf life of the lyophilized composition is about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 10 hours, about 15 hours, about 20 hours, or about 24 hours. In some embodiments, the shelf life of the lyophilized composition is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 10 days, about 15 days, about 20 days, about 25 days, About 28 days, about 30 days, or about 31 days. In some embodiments, the shelf life of the lyophilized composition is about 1 month, about 2 months, about 3 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months. In some embodiments, the shelf life of the lyophilized composition is about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years. In some embodiments, the shelf life of the lyophilized composition is about 0.5 minutes to about 60 minutes, about 1 minute to about 60 minutes, about 2 minutes to about 60 minutes, about 3 minutes to about 60 minutes, about 4 minutes to About 60 minutes, about 5 minutes to about 60 minutes, about 10 minutes to about 60 minutes, about 15 minutes to about 60 minutes, about 20 minutes to about 60 minutes, about 30 minutes to about 60 minutes, or about 45 minutes to about 60 Within minutes. In some embodiments, the shelf life of the lyophilized composition is about 1 hour to about 24 hours, about 2 hours to about 24 hours, about 3 hours to about 24 hours, about 4 hours to about 24 hours, about 5 hours to Within a range of about 24 hours, about 10 hours to about 24 hours, about 15 hours to about 24 hours, or about 20 hours to about 24 hours. In some embodiments, the shelf life of the lyophilized composition is about 1 day to about 31 days, 2 days to about 31 days, 3 days to about 31 days, 4 days to about 31 days, 5 days to about 31 days, 10 days to about 31 days, 15 days to about 31 days, 20 days to about 31 days, 25 days to about 31 days. In some embodiments, the shelf life of the lyophilized composition is about 1 month to about 12 months, 2 months to about 12 months, 3 months to about 12 months, 4 months to about 12 months , 5 months to about 12 months, 6 months to about 12 months, 7 months to about 12 months, 8 months to about 12 months, 9 months to about 12 months, 10 months to In the range of about 12 months or about 11 months to about 12 months. In some embodiments, the shelf life of the lyophilized composition is about 1 year to about 2 years, about 1 year to about 3 years, about 1 year to about 4 years, about 1 year to about 5 years, about 2 years to About 3 years, about 2 years to about 4 years, about 2 years to about 5 years, about 3 years to about 4 years, about 3 years to about 5 years, or about 4 years to about 5 years.

In some embodiments, the shelf life of the lyophilized pharmaceutical composition can be increased by about 10% to about 300%, about 20% to about 300%, about 50% to about 300%, compared to the pharmaceutical composition that has not been lyophilized. About 100% to about 300%, about 150% to about 300%, about 200% to about 300%, about 250% to about 300%, about 10% to about 250%, about 20% to about 250%, about 50 % To about 250%, about 100% to about 250%, about 150% to about 250%, about 200% to about 250%, about 10% to about 200%, about 20% to about 200%, about 50% to About 200%, about 100% to about 200%, about 150% to about 200%, 10% to about 150%, 20% to about 150%, 50% to about 150%, 100% to about 150%, 10% To about 100%, 20% to about 100%, 50% to about 100%, 10% to about 50%, 20% to about 50%, 10% to about 20%, at least about 1%, at least about 2%, At least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, At least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, At least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%. In some embodiments, the shelf life of the lyophilized pharmaceutical composition can be increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to About 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 Times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, At least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times. Depending on the particular application, one or more components of the composition of the invention may be lyophilized. In some embodiments, the composition of the invention comprises at least one lyophilized peptide. In some embodiments, the composition of the present invention comprises at least one lyophilized peptide and a lyophilized pH adjusting agent. One or more lyophilized components can be combined with the sterile solution prior to administration. In some embodiments, at least one lyophilized peptide can be combined with a non-lyophilized pharmaceutically acceptable carrier prior to administration. In some embodiments, at least one lyophilized peptide can be combined with a non-lyophilized pH adjusting agent and a non-lyophilized pharmaceutically acceptable carrier before administration. In some embodiments, at least one lyophilized peptide can be combined with a non-lyophilized pH adjusting agent, a non-lyophilized immunomodulator or adjuvant, and a non-lyophilized pharmaceutically acceptable carrier before administration.

The invention also provides a method for preparing a pharmaceutical composition. The method of preparing the composition of the present invention may comprise combining at least one peptide with a pH adjusting agent and a pharmaceutically acceptable carrier. The method of preparing the composition of the present invention may comprise combining at least one peptide with a pH adjusting agent, an immunomodulating agent or an adjuvant, and a pharmaceutically acceptable carrier. The method of preparing the composition of the present invention may also include combining the peptide with the pH adjusting agent and a pharmaceutically acceptable carrier so that the concentration of the pH adjusting agent is less than 1.0 mM. The method of preparing the composition of the present invention may also comprise combining the peptide with a pH adjusting agent, an immunomodulating agent or an adjuvant and a pharmaceutically acceptable carrier such that the concentration of the pH adjusting agent is less than 1.0 mM. The method of preparing the pharmaceutical composition may include preparing a solution containing at least one neoantigenic peptide. The preparation of such a solution may refer to dissolving at least one peptide in a solvent; for example, dissolving the peptide in DMSO to prepare a 50 mg/ml peptide in the DMSO stock solution. Steps or procedures commonly used in the treatment of solutions may include agitation, mixing, shaking, sonic treatment, heating, cooling, dilution, filtration, etc., and those skilled in the art can generally identify suitable steps or procedures and for specific peptides or solutions When additional steps or procedures are required. There are various possible orders for combining the components of the composition of the present invention, and the present invention covers all orders. In some embodiments, at least one peptide is first dissolved in DMSO, and the DMSO stock solution is combined with an aqueous solution containing a pH adjusting agent. In other embodiments, at least one peptide and a pH adjusting agent are simultaneously dissolved in the solution. In some embodiments, one or more filtration steps may be employed to remove any insoluble materials before and/or after any combination of the components of the composition of the invention.

Similarly, provided herein are methods for preparing new antigen peptide solutions. The preparation of such a solution may refer to dissolving at least one peptide in a solvent; for example, dissolving the peptide in DMSO to prepare a 50 mg/ml peptide stock solution in DMSO. The method of preparing the solution of the present invention may also include combining the peptide and the pH adjusting agent so that the concentration of the pH adjusting agent is less than 1.0 mM. There are various possible sequences for combining the components of the solution of the invention, and the invention covers all sequences. In some embodiments, at least one peptide is first dissolved in DMSO, and the DMSO stock solution is combined with an aqueous solution containing a pH adjusting agent. In other embodiments, at least one peptide and a pH adjusting agent are simultaneously dissolved in the solution. In some embodiments, one or more filtration steps may be employed to remove any insoluble materials before and/or after any combination of the components of the composition of the invention. Vaccines and kits

In some aspects, provided herein is a vaccine or immunogenic composition comprising a pharmaceutical composition. In some embodiments, the vaccine is a neoplastic vaccine. In some aspects, a kit comprising one or more components of the formulation is provided. The kit may include instructions to provide the user with information such as how to use the kit and how to administer the resulting formulation. If applicable, each component of the composition of the present invention may be included in the kit in the form of a liquid, a solid, or a combination thereof; for example, at least one peptide and/or pH adjusting agent may be pre-dissolved in the kit Depending on the specific instructions provided by the kit, the solution, or it may be in solid form, before administration, the user may need to combine it with the liquid. In some embodiments, the kit includes a lyophilized composition that includes at least one neoantigenic peptide. In some embodiments, the kit may include instructions that teach the user to combine at least one peptide with a pH adjusting agent so that the pH adjusting agent is present in the resulting solution at a concentration of less than 1.0 mM. In some embodiments, the kit may further comprise a pharmaceutically acceptable carrier, such as water, DMSO, other (co)solvents, or a combination thereof. Depending on the pharmaceutical composition or vaccine composition, application, administration route and other considerations, the kit may contain various containers (such as vials, bottles, tubes, bags and a piece of (non) soluble paper), various liquids (such as solvents , Co-solvents and buffers) and various solids (such as salts, sugars and lyophilized peptides). In some embodiments, the kit contains a single dose or multiple doses that do not need to be restored, wherein the user can administer the formulation contained in the kit as is. In other embodiments, the kit contains a single dose or multiple doses that need to be restored, where the user can combine the components provided in the kit with other components that may or may not be provided in the kit; for example The user may need to combine the lyophilized peptide provided in the kit with the water or buffer that may or may not be provided in the kit.

The kits provided herein may contain viral vectors. In the presence of viral vectors, at least one neoantigen peptide can be encoded and expressed in cells in vivo or in vitro. Common viral vectors that can be used include lentivirus-based vectors, adenovirus-based vectors, adeno-associated virus (AAV)-based vectors, based on viruses such as vaccinia virus, ALVAC, Modified Vaccinia Ankara (MVA) virus And modified vaccinia virus of Copenhagen strain (vaccinia virus) vectors; and based on such as murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV) and human immunity Vectors of retroviruses for deficiency virus (HIV).

The invention also provides a method for preparing a vaccine composition. The method of preparing a vaccine composition may include preparing a solution containing at least one neoantigenic peptide. The preparation of such a solution may refer to dissolving at least one peptide in a solvent; for example, dissolving the peptide in DMSO to prepare a 50 mg/ml peptide in the DMSO stock solution. The method of preparing a vaccine composition may include combining the peptide with a pH adjusting agent and a pharmaceutically acceptable carrier so that the concentration of the pH adjusting agent is less than 1.0 mM. The method of preparing a vaccine composition may also include combining at least one peptide with an immunomodulator or adjuvant. There are various possible sequences for combining the components of the vaccine composition of the present invention, and the present invention covers all sequences. In some embodiments, at least one peptide is first dissolved in DMSO; then the DMSO stock solution is combined with an aqueous solution containing a pH adjusting agent; and finally the solution containing an immunomodulating agent or adjuvant is combined into the above peptide-pH adjustment Agent solution. In other embodiments, at least one peptide, pH adjuster, and immunomodulator or adjuvant are simultaneously dissolved in the solution. In some embodiments, one or more filtration steps may be employed to remove any insoluble materials before and/or after any combination of the components of the composition of the invention. Therapeutic treatment

Also encompassed is a method of treating a condition or disease by administering the compositions provided herein. In some embodiments, the condition or disease is a neoplasm. In some embodiments, the neoplasm is cancer. A method of treating a subject suffering from a neoplasm may refer to inducing an immune response in the subject and treating the neoplasm of the subject by administering a vaccine or pharmaceutical composition to the subject. The composition can be used in subjects who have been diagnosed with or are at risk of developing neoplasia. The composition may be administered to the subject in an amount and time interval appropriate to the subject (in the case of a series of administrations). For example, a method of treating a condition or disease may include administering a unit dose of the composition to the subject at regular intervals, such as one day, one week, and one month. In some embodiments, the method comprises administering to the subject the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more compositions, wherein the independent Choose each investment (including composition, amount, and timing).

In some embodiments, specific examples of cancers that can be prevented or treated according to the composition include, but are not limited to: kidney cancer (kidney cancer), kidney cancer (kidney cancer), glioblastoma multiforme, metastatic breast cancer; Breast cancer; Breast sarcoma; Neurofibroma; Neurofibromatosis; Pediatric tumor; Neuroblastoma; Malignant melanoma; Epidermal carcinoma; Leukemia, such as but not limited to acute leukemia, acute lymphocytic Leukemia, acute myelogenous leukemia (such as myeloblastic, promyelocytic, myelomonocytic, mononuclear, erythroleukemia, and spinal dysplasia syndrome), chronic leukemia (such as but not limited to chronic myeloid cells (Granulococcal leukemia, chronic lymphocytic leukemia, hairy cell leukemia); polycythemia vera; lymphomas, such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myeloma, such as But not limited to smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; Undetermined importance of single globulinosis; benign single globulinosis; heavy chain disease; bone cancer and connective tissue sarcoma, such as but not limited to bone sarcoma, myeloma bone disease, multiple myeloma, cholesteatoma Skeletal osteosarcoma, Paget's disease of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, skeletal fibrosarcoma, chordoma, periosteal sarcoma, soft tissue sarcoma, blood vessels Sarcomas (angiosarcoma/hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors, such as but not limited to glioma Tumors, astrocytomas, brainstem gliomas, ependymomas, oligodendrogliomas, nonglial tumors, auditory schwannomas, craniopharyngiomas, neural tube blastomas, ridges Meningioma, pineal gland cell tumor, pineal gland cell tumor and primary brain lymphoma; breast cancer, including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, Mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease (including juvenile Paget's disease) and inflammatory breast cancer; adrenal cancers such as but not limited to pheochromocytoma and adrenal cortical cancer Thyroid cancer, such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and pleomorphic thyroid cancer; pancreatic cancer, such as but not limited to insulinoma, gastrinoma, glucagonoma, vasoactive intestine Peptide tumors, somatostatin secreting tumors and carcinoid or islet cell tumors; pituitary cancers, such as but not limited to Cushing's disease, Prolactin secreting tumors, acromegaly, and diabetic diabetes insipidus; eye cancers, such as but not limited to ocular melanomas (such as iris melanoma, choroidal melanoma, and ciliary body melanoma) and retinoblastoma; vaginal cancer , Such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancer, such as but Not limited to squamous cell carcinoma and adenocarcinoma; uterine cancer, such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancer, such as but not limited to ovarian epithelial carcinoma, borderline tumor, germ cell tumor and stromal tumor; Cervical carcinoma; esophageal cancer, such as but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma And oat cell (small cell) carcinoma; gastric cancer, such as but not limited to adenocarcinoma, mycosis (polyposis), ulcer type, superficial spread, diffuse spread, malignant lymphoma, liposarcoma, fibrosarcoma and carcinosarcoma; Colon cancer; KRAS mutation colorectal cancer; colon cancer; rectal cancer; liver cancer, such as but not limited to hepatocellular carcinoma and hepatoblastoma; gallbladder cancer, such as adenocarcinoma; cholangiocarcinoma, such as but not limited to papillary , Nodules and scattered; lung cancer, such as KRAS mutation non-small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma and small cell lung cancer; lung cancer; Testicular cancer, such as but not limited to germ cell tumor, sperm cell carcinoma, pleomorphism, classic/typical, spermatocyte type, non-seminoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma (Yolk sac tumor); prostate cancer, such as but not limited to non-androgen-dependent prostate cancer, androgen-dependent prostate cancer, adenocarcinoma, leiomyosarcoma and rhabdomyosarcoma; penile cancer; oral cancer, such as but not limited to squamous Cell carcinoma; Basal cell carcinoma; Salivary adenocarcinoma, such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; Pharyngeal carcinoma, such as but not limited to squamous cell carcinoma and verrucous; skin cancer, Such as but not limited to basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, freckle malignant melanoma, acral freckle nevus melanoma; kidney cancer, such as but not Limited to renal cell carcinoma, adenocarcinoma, adrenal tumor, fibrosarcoma, transitional cell carcinoma (renal pelvis and/or ureter); renal carcinoma; Wilms' tumor; bladder cancer, such as but not limited to transition Cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endothelial sarcoma, lymphatic endothelial sarcoma, mesothelioma, synovial tumor, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland Carcinoma, papillary carcinoma and papillary adenocarcinoma.

In some embodiments, the composition may be administered to a subject suffering from neoplasia immediately after preparing the composition. In some embodiments, the composition may be about 0.5 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes after preparing the composition , About 30 minutes, about 45 minutes, or about 60 minutes of administration to subjects with neoplasia. In some embodiments, the composition may be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 10 hours, about 15 hours, about 20 hours, or about 24 hours after preparing the composition Administration to subjects with neoplasia. In some embodiments, the composition may be about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 10 days, about 15 days, about 20 days, about 25 days after preparing the composition , About 28 days, about 30 days, or about 31 days to administer to subjects with neoplasia. In some embodiments, the composition may be about 1 month, about 2 months, about 3 months, about 5 months, about 6 months, about 7 months, about 8 months, Subjects with neoplasia are administered for about 9 months, about 10 months, about 11 months, or about 12 months. In some embodiments, the composition may be administered to a subject with neoplasia about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years after the composition is prepared. In some embodiments, the composition may be about 0.5 minutes to about 60 minutes, 1 minute to about 60 minutes, 2 minutes to about 60 minutes, 3 minutes to about 60 minutes, 4 minutes to about 60 minutes after preparing the composition , 5 minutes to about 60 minutes, 10 minutes to about 60 minutes, 15 minutes to about 60 minutes, 20 minutes to about 60 minutes, 30 minutes to about 60 minutes, or 45 minutes to about 60 minutes. Subject administered. In some embodiments, the composition may be about 1 hour to about 24 hours, 2 hours to about 24 hours, 3 hours to about 24 hours, 4 hours to about 24 hours, 5 hours to about 24 hours after preparing the composition , 10 hours to about 24 hours, 15 hours to about 24 hours, or 20 hours to about 24 hours in the range of administration to subjects with neoplasia. In some embodiments, the composition may be about 1 day to about 31 days, 2 days to about 31 days, 3 days to about 31 days, 4 days to about 31 days, 5 days to about 31 days after preparing the composition , 10 days to about 31 days, 15 days to about 31 days, 20 days to about 31 days, 25 days to about 31 days of administration to subjects with neoplasia. In some embodiments, the composition may be about 1 month to about 12 months, 2 months to about 12 months, 3 months to about 12 months, 4 months to about 12 after the composition is prepared Month, 5 months to about 12 months, 6 months to about 12 months, 7 months to about 12 months, 8 months to about 12 months, 9 months to about 12 months, 10 months Subjects with neoplasia are administered within a range of about 12 months or about 11 months to about 12 months. In some embodiments, the composition may be about 1 year to about 2 years, about 1 year to about 3 years, about 1 year to about 4 years, about 1 year to about 5 years, about 2 years after the composition is prepared Up to about 3 years, about 2 years to about 4 years, about 2 years to about 5 years, about 3 years to about 4 years, about 3 years to about 5 years, or about 4 years to about 5 years The subject of the tumor is administered.

The invention covers a pharmaceutical or vaccine formulation, which is used to prepare a medicament for treating a condition or disease. In some embodiments, the medicament may comprise one or more lyophilized or non-lyophilized components of the composition of the invention or a combination or isolate thereof. In addition, the present invention provides a method for preparing a medicament containing the composition of the present invention. In some embodiments, the condition or disease is a neoplasm. Examples

The present invention is more precisely illustrated by the following examples. However, it should be understood that the present invention is not limited by these examples in any way. Example 1 : Peptide formulation and solubility

方程式 2 ： 肽含量= 100 - %TFA - %水，其中水= 6.45方程式 3 ：

方程式 4 ：

The peptide is synthesized chemically and purified. All peptides were weighed in 5 mL Eppendorf tubes using the XP105 Delta Range balance from Mettler Toledo. The theoretical peptide content of the lyophilized peptide is determined by weight, theoretical trifluoroacetic acid (TFA) content, and percent purity. The percentage peptide content is determined using equations 1 and 2; alternatively, it can be determined experimentally. The total target weight (mg) was then calculated using Equation 3, and Equation 4 was used to determine the volume of DMSO required to obtain a peptide concentration of 50 mg/ml. Equation 1 :

Equation 2 : Peptide content = 100-%TFA-% water, where water = 6.45 Equation 3 :

Equation 4 :

The succinate buffer used in this study was prepared daily by diluting 1 M sodium succinate in 5% dextrose in water (D5W) to the desired concentration. After the peptide had been dissolved in DMSO to obtain a 50 mg/ml stock solution, 60 μL of the stock solution was then used for dilution with 1.44 mL of 5 mM, 0.75 mM, 0.5 mM, or 0.25 mM sodium succinate in D5W. The pH of each peptide solution was measured using a Gene Mate meter with electrode pHE-11. The peptide solution was then stored under ambient conditions for a minimum of 2 hours to allow any potential precipitation or gel to appear. If the solution appeared transparent and no gel was formed during the study, the peptide was considered to be completely soluble.

Twenty peptides previously determined to be partially soluble or insoluble in formulations containing approximately 5.0 mM succinate were tested in formulations containing lower sodium succinate concentrations (see Table 5). Another 20 peptides soluble in formulations containing approximately 5.0 mM succinate were also tested at lower succinate conditions to confirm that their solubility was not adversely affected by the reduction of succinate concentration (see Table 4 ). The calculated physical properties of the peptides used herein include hydrophobicity (by Kate-Dulitt method or by calculating its HYDRO value) and pI, and are shown in Tables 4 and 5. Test peptides with a range of pI and hydrophobicity values to better understand how their values affect solubility within the succinate concentration range.

All twenty peptides soluble in formulations containing approximately 5.0 mM succinate remained soluble at lower succinate concentrations (Table 4, see Figure 1 for images). Lowering the pH adjuster concentration to less than 1.0 mM does not adversely affect the solubility of peptides soluble at higher pH adjuster concentrations.

Table 4 : Solubility results for 20 formulations of peptides soluble in 5 mM succinate and remaining soluble in 0.75 mM, 0.50 mM, and 0.25 mM succinate.

Fourteen of the twenty peptides insoluble in formulations containing approximately 5.0 mM succinate are soluble in 0.75 mM, 0.5 mM, and 0.25 mM succinate, but three are only soluble in 0.25 mM Succinate (Table 5, see Figure 2 for images). Therefore, "save" at least 70% of the peptides by using a formulation with a pH adjuster concentration of less than 1.0 mM instead of a pH adjuster concentration of 5.0 mM. Of the six peptides that remain insoluble, four of them have a pI of 4.3, while the other two have a neutral pI and higher hydrophobicity. No peptides with a pi of about 4 and insoluble in formulations containing about 5.0 mM succinate were "rescued" by lower succinate concentrations. Considering that the calculated pI values of these peptides are similar to the pH of the formulation, this observation is not surprising. Several soluble peptides with a pi equal to or lower than 4.3 have also been tested and remain soluble in lower succinate concentrations despite having a lower pH. In general, reducing the concentration of the pH adjusting agent in the compositions provided herein increases the solubility of the peptide.

*?表明在無過濾回收研究情況下不能確定肽是否可溶或不可溶。實例 2 ： 藉由濁度確定肽溶解 Table 5 : Solubility results for 20 formulations of peptides that are insoluble in 5 mM succinate and become soluble in 0.75 mM, 0.50 mM, and 0.25 mM succinate.

*? indicates that it is not possible to determine whether the peptide is soluble or insoluble without filtration and recovery studies. Example 2 : Determination of peptide dissolution by turbidity

Whether the peptide or peptide mixture is completely soluble in the formulation at a given temperature can be determined using turbidity measurements. The method outlined in Example 1 should be used to prepare pharmaceutical formulations containing peptides or peptide mixtures. At the same time, the composition of the reference formulation is approximately the same as the pharmaceutical formulation except for the lack of the peptide or peptide mixture to be prepared. Subsequent reference and pharmaceutical formulation turbidity should be measured using any instrument or technique suitable for measuring turbidity. Such instruments and techniques include but are not limited to visual inspection of formulations, turbidity meters, or turbidity sensors. We expect that when the peptide or peptide mixture is soluble in the pharmaceutical formulation, the turbidity should not increase compared to the reference formulation. We expect that when the peptide or peptide mixture is partially soluble or insoluble in the pharmaceutical formulation, the turbidity should be increased compared to the reference formulation. Example 3 : Formulation with poly - ICLC

Example 3 demonstrates that the composition of the present invention is compatible with poly-ICLC (poly ICLC) (an RNA-based immunostimulant). Although a lower succinate concentration is a suitable universal formulation for peptide solubility, it is assumed that it may not be compatible with poly-ICLC because the peptide in the lower succinate concentration has a pH of 3.2 to 4.0 and Poly-ICLC may precipitate when combined with lower pH peptide solutions. The approximate pKa of the phosphate group in the RNA is 2 and the polyICLC already exists in the form of a cloudy suspension; therefore, it is possible that the lower pH of the formulation may cause the polyICLC to further precipitate or aggregate. Therefore, polyICLC was combined with various peptides in 5 mM, 0.50 mM, and 0.25 mM succinate to demonstrate that poly-ICLC is compatible with the formulations of the present invention that include a pH adjuster at a concentration of less than 1.0 mM.

All pH values for this experiment were measured with Mettler Toledo's pH/ion laboratory bench gauge S220-B and pH inLab microprobes, which were calibrated with pH 4.0, 7.0 and 10.0 standards immediately before use. All samples were stored at room temperature after being combined with poly-ICLC so that all possible precipitation appeared. Warm the peptide and poly-ICLC to room temperature before use. During this time a 50 mg/ml DMSO stock solution was prepared as described above and diluted to 2 mg/ml by adding 10 μL peptide stock solution to 240 μL succinate buffer in D5W. Collect peptides by combining 100 μL of each of the 5 specified peptides. The aggregate and poly-ICLC were mixed at a ratio of peptide aggregate: poly-ICLC 2:1 (200 μL peptide combination to 100 μL poly-ICLC).

A comparison was made between a combination of peptides soluble in 5 mM succinate and a combination of the same peptide soluble in 0.5 mM succinate and 0.25 mM succinate. The pH values of the individual and combined peptides were measured before they were mixed with poly-ICLC in the ratio of peptide aggregate: poly-ICLC 2:1. The resulting solution had a similar appearance to a cloudy suspension (see Figure 3 for an image), and no increase in precipitation occurred when poly-ICLC was combined with various peptide aggregates. Importantly, peptide aggregates insoluble in formulations containing about 5.0 mM succinate were also tested at 0.25 mM and 0.5 mM succinate and remained soluble when combined with poly-ICLC. In addition, for 0.25 mM succinate the pH consistently reached approximately 4.80 and for 0.5 mM succinate approximately 5.20, both values were within the range necessary for subcutaneous injection (Table 6). Example 2 demonstrates that using a sodium succinate concentration equal to or lower than 0.75 mM will maximize the number of peptides that are qualified for solubility while remaining compatible with poly-ICLC.

*聚-ICLC之pH量測為7.23實例 4 ：合成 GATA3 neoORF 肽之溶解度試驗 Table 6 : pH values of individual and combined peptides soluble in 5 mM, 0.5 mM and 0.25 mM succinate

*The pH measurement of poly-ICLC is 7.23 Example 4 : Solubility test of synthetic GATA3 neoORF peptide

The solubility of each peptide in Table 7 below was tested in various specified solutions. SS = sodium succinate hexahydrate. Formulation A tested included 4% DMSO, 5 mM sodium succinate hexahydrate (SS)/D5W. Formulation B tested did not include DMSO, including 5 mM SS/D5W. Formulation C tested did not include DMSO, including 0.25 mM SS/D5W. The synthesis of 33mer L15 containing two cysteines was carried out using pseudoproline dipeptide building blocks and combining ES and AT into the sequence. Pseudoproline increases the purity of the crude product of L15 by preventing aggregation during solid-phase peptide synthesis, which allows L15 to be purified to 95% purity.

實例 5 - 設計用於向受試者投與之合成 GATA3 neoORF 肽之集合體 Table 7 : Peptide solubility

Example 5 - An assembly designed to synthesize GATA3 neoORF peptides for administration to subjects

實例 6 - GATA3 neoORF 肽合成 The collection system of various designated GATA3 peptides is designed according to Table 8 below. For example, "Design 1" contains three peptide aggregates, where aggregate 1 contains three peptides (that is, L7, L8, and L14), and aggregate 2 contains two peptides (that is, L9 and L10c) and aggregate 3 Contains two peptides (that is, L15 and L11f). For example, "Design 6" contains two peptide aggregates, where aggregate 1 contains four peptides (ie, L7, L8, L9, and L14) and aggregate 2 contains two peptides (ie, L15 and L11f). For example, "Design 10" contains four peptide aggregates, in which aggregate 1 contains five peptides (that is, L7, L8, L9, L10c, and L14), and aggregate 2 contains one peptide (that is, L15), and Body 3 contains a peptide (ie L11f) and assembly 4 contains a peptide (ie L11i). The concentration of each peptide in the pool can be changed according to those skilled in the preparation of peptide formulations. Table 8 : Description of GATA3 aggregate design

Example 6 - GATA3 neoORF peptide synthesis

Conventional synthesis was performed with 650 mg of crude material target. The following Fmoc-amino acids with appropriate side chain protection are used to construct the L7 peptide (EPCSMLTGPPARVPAVPFDLH (SEQ ID NO: 42)): Fmoc-Ala-OH∙H ₂ O, Fmoc-Cys(Trt)-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Thr(tBu)-OH and Fmoc-Val-OH. The C-terminal histidine is incorporated into the sequence by using H-His(Trt)-2Cl-Trt resin or Fmoc-His(Trt)-Wang (LL) resin preloaded on the resin. Fmoc-Asp(OMpe)-OH can be used instead of Fmoc-Asp(OtBu)-OH to help improve the synthesis, such as the combination of sequences with "DG" to minimize asparagine formation.

The peptide sequence was swollen with dimethylformamide (DMF) and drained twice. The synthesis was started by using 20% piperidine in DMF and mixing with nitrogen to mix to deprotect the N-α-FMOC protecting group. After draining, the resin was washed with DMF. Next, 6 mL of 0.4 M amino acid solution and 5.8 mL of 0.4 M HCTU and 6 mL of 0.8 M DIEA were added over 20 minutes. The coupling reaction is performed with nitrogen-dispensing mixing, and then the reaction vessel (RV) is drained. Repeat the amino acid, HCTU, and DIEA additions for the second coupling cycle with the same mixing and draining parameters as the first coupling step. The resin was then washed again with DMF. This cycle is repeated for each amino acid residue. The final deprotection method removes the N-terminal Fmoc via 20% piperidine in DMF, and the resin is washed with DMF and then MeOH. The resin was removed under nitrogen on the instrument.

For microwave synthesis, the same Fmoc-amino acid starting material is used, in which only the Fmoc-His(Trt)-Wang (LL) resin (not the H-His(Trt)-2ClTrt resin) is used to incorporate the C-terminal Histidine.

On the microwave synthesizer, the resin was swelled in DMF until it was transferred to the microwave reaction vessel (RV) through the HT line. While in RV, the Fmoc-His(Trt)-OH supported resin was treated with 25% pyrrolidine in DMF at 85°C/90W followed by 100°C/20W to remove N-α-FMOC. Next, the RV was drained and washed with DMF, and drained again. Stylized Fmoc-amino acid (0.5 M in DMF) and 4 M DIC and 0.25 M Oxymapure were added to the RV. This coupling reaction is followed by heating at 105°C/288W, then heating at 105°C/73W. This first deprotection is first diluted with DMF, however any subsequent deprotection does not require this step because RV already contains DMF from the coupling reaction. The deprotection, washing and coupling cycles are repeated for each residue until the peptide has been synthesized. For arginine residues, a second coupling step is performed, where the solution is drained after a single coupling is performed, and the coupling step is repeated before proceeding to deprotection. The final deprotection of the N-terminal Fmoc group was performed as above except that it was drained and washed twice with DMF before being transferred back to the original HT resin location via DMF.

After synthesis, the resin was transferred to a sintered syringe using DMF, rinsed with MeOH and dried using a vacuum header. Then use reagent K (82.5% trifluoroacetic acid (TFA), 5% water, 5% thioanisole, 5% phenol and 2.5% ethanedithiol) at room temperature using a vertical holder on the vibrating oscillator Continue for three hours to split the resin.

The split mixture is then dispensed into cold ether or cold methyl tert-butyl ether (MTBE) via a sintered filter syringe. Subsequently, each syringe was rinsed with 95:5 trifluoroacetic acid:water solution by stirring. The rinse solution is then added to the rest of the mixture/ether mixture. The mixture is then centrifuged. After decanting the ether, another cold ether wash was added. The container was vortexed and centrifuged again. Repeat this to fully rinse the pellets. Decant the final wash and dry the pellets via a vacuum dryer. The pellet samples were dissolved in solvents (eg DMSO, DMF, water or acetonitrile) and analyzed via UPLC-MS for identity, crude product purity and residence time. In a similar manner, other peptides are made using amino acids and preloaded resins specific to their sequences, such as L14 (SMLTGPPARVPAVPFDLH (SEQ ID NO: 43)), L8 (GPPARVPAVPFDLHFCRSSIMKPKRD (SEQ ID NO: 44)), L10c (KPKRDGYMFLKAESKI (SEQ ID NO: 59)), L11h (FLKAESKIMFATLQR (SEQ ID NO: 63)) and L11i (ESKIMFATLQRSSL (SEQ ID NO: 64)). Example 7 - GATA3 neoORF peptide synthesis

The following Fmoc-amino acids are used to synthesize peptide L15 (KPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH (SEQ ID NO: 67)): Fmoc-Ala-OH∙H ₂ O, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc- Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc -Trp(Boc)-OH and Fmoc-Tyr(tBu)-OH. The C-terminal histidine is incorporated into the sequence by using H-His(Trt)-2Cl-Trt resin or Fmoc-His(Trt)-Wang (LL) resin preloaded on the resin. Fmoc-Asp(OMpe)-OH can be used instead of Fmoc-Asp(OtBu)-OH to help improve the synthesis, such as the combination of sequences with "DG" to minimize asparagine formation. When serine (Ser, S) and threonine (Thr, T) residues are present, amino acid dipeptides (pseudoproline) are incorporated to increase synthetic yields, such as Fmoc-Ser(tBu)- Ser(psi(Me,Me)pro)-OH replaces "SS", Fmoc-Ala-Thr(psi(Me,Me)pro)-OH replaces "AT" and Fmoc-Glu(OtBu)-Ser(psi(Me ,Me)pro)-OH instead of "ES". For some L15 synthesis, use all three pseudoproline (ie Fmoc-Ser(tBu)-Ser(psi(Me,Me)pro)-OH instead of "SS", Fmoc-Ala-Thr(psi(Me,Me )pro)-OH instead of "AT" and Fmoc-Glu(OtBu)-Ser(psi(Me,Me)pro)-OH) instead of "ES"). For other L15 synthesis, the following pseudoproline and pseudoproline combination are used: Fmoc-Ala-Thr(psi(Me,Me)pro)-OH instead of "AT" and Fmoc-Glu(OtBu)-Ser(psi (Me,Me)pro)-OH replaces "ES"; Fmoc-Ser(tBu)-Ser(psi(Me,Me)pro)-OH replaces "SS" and Fmoc-Glu(OtBu)-Ser(psi(Me ,Me)pro)-OH replaces "ES"; Fmoc-Ser(tBu)-Ser(psi(Me,Me)pro)-OH replaces "SS" and Fmoc-Ala-Thr(psi(Me,Me)pro) -OH replaces "AT"; Fmoc-Ala-Thr(psi(Me,Me)pro)-OH replaces "AT"; Fmoc-Glu(OtBu)-Ser(psi(Me,Me)pro)-OH) replaces "ES"; and Fmoc-Ser(tBu)-Ser(psi(Me,Me)pro)-OH instead of "SS".

The peptide sequence was swollen with DMF and drained twice. The synthesis was started by using 20% piperidine in DMF to mix with nitrogen to mix to deprotect the N-α-FMOC group. After draining, the resin was washed with DMF. Next, 6 mL of 0.4 M amino acid solution and 5.8 mL of 0.4 M HCTU and 6 mL of 0.8 M DIEA were added. The coupling reaction was carried out with nitrogen-dispensing mixing and then the reaction vessel (RV) was drained. Repeat the amino acid, HCTU, and DIEA additions for the second coupling cycle with the same mixing and draining parameters as the first coupling step. The resin was then washed again with DMF. This cycle is repeated for each amino acid residue. The final deprotection method removes the N-terminal Fmoc via 20% piperidine in DMF, and the resin is washed with DMF and then MeOH. The resin was covered on the instrument under nitrogen until it was removed. For microwave synthesis, the same amino acid starting material is used, where only Fmoc-His(Trt)-Wang (LL) resin (not H-His(Trt)-2ClTrt resin) is used to incorporate C-terminal histamine acid.

On the microwave synthesizer, the resin was swelled in DMF until it was transferred to the microwave reaction vessel (RV) through the HT line. While in RV, Fmoc-His(Trt) loaded resin was treated with 25% pyrrolidine in DMF at 85°C/90W followed by 100°C/20W to remove N-α-FMOC. This first deprotection is first diluted with DMF; however, any subsequent deprotection does not require this step because RV already contains DMF from the coupling reaction. The RV was then drained and washed with DMF, and drained again. The stylized Fmoc-amino acid (0.5 M in DMF) and 4 M DIC and 0.25 M Oxymapure were then added to the RV. This coupling reaction is followed by heating at 105°C/288W, then heating at 105°C/73W. The deprotection, washing and coupling cycles are repeated for each residue until the peptide has been synthesized. For arginine residues, there is a second coupling step, where the solution is drained after a single coupling is performed, and the coupling step is repeated before proceeding to deprotection. The final deprotection of the N-terminal Fmoc group was performed in the same way as all other deprotection steps except that it was drained and washed twice with DMF before being transferred back to the original HT resin location via DMF.

After synthesis, the resin was transferred to a sintered syringe using DMF, rinsed with MeOH and dried using a vacuum header. Then use reagent K (82.5% trifluoroacetic acid (TFA), 5% water, 5% thioanisole, 5% phenol and 2.5% ethanedithiol) at room temperature using a vertical holder on the vibrating oscillator Split the resin.

The split mixture is then dispensed into cold ether (or cold MTBE) via a sintered filter syringe. Subsequently, each syringe was rinsed with 95:5 trifluoroacetic acid:water solution by stirring. The rinse solution is then added to the rest of the mixture/ether mixture. The mixture is then centrifuged. After decanting the ether, another cold ether wash was added. The container was vortexed and centrifuged again. Repeat this to fully rinse the pellets. Decant the final wash and dry the pellets via a vacuum dryer. The pellet samples were dissolved in solvents (eg DMSO, DMF, water or acetonitrile) and analyzed via UPLC-MS for identity, crude product purity and residence time.

Use amino acids and preloaded resins and pseudoproline derivatives specific for their sequences in a similar manner (when serine (Ser, S) or threonine (Thr, T) residues are present) And Fmoc-Asp(OMpe)-OH as described above makes other peptides, such as L9 (LHFCRSSIMKPKRDGYMFLKAESKI (SEQ ID NO: 45)). Example 8 - Solubility study

First, the solubility of various GATA3 peptides with new epitopes was tested using 5 mM sodium succinate (SS) in D5W containing 4% DMSO. Based on the initial results, the formulation strategy was improved by adjusting the concentration of sodium succinate (SS) and the amount of DMSO, which led to the selection of seven peptides. The aggregation strategy of these peptides was determined for solubility and compatibility with polyICLC. Based on these results, three aggregates are selected. Two of the 0.25 mM SS/D5W each have an aggregate of only one peptide and a third aggregate of 5 peptides in 5 mM SS/D5W. After combining with the polyICLC, the pH of the aggregate was all pH 5.0 to 6.0 and there was very little loss during filtration.

All peptides screened for the following studies are listed in Table 9.

Table 9 Peptide sequences

All buffers are prepared daily. D5W is prepared by weighing dextrose and adding ultrapure water to dextrose to achieve the appropriate volume. For example, add water to 12.5 g dextrose to reach a total volume of 250 mL. To prepare 50 mL of 5 mM SS/D5W by weighing 67.54 mg SS was weighed and added to D5W to reach a total volume of 50 mL. To prepare 0.25 mM SS/D5W, dilute 2.5 mL 5 mM SS/D5W with 47.5 mL D5W.

The% peptide content of each peptide is determined as follows: The total theoretical TFA is equal to the sum of the number of positive charges (N-terminal, Arg, Lys, and His). Enter that number into the following equation, where MW is the molecular weight of the peptide: Equation 1.% TFA = 100 × ((%TFA× 114.02)/((%TFA× 114.02) + MW))

This value is then used to calculate the percentage peptide content using 6.45% as the theoretical water content: Equation 2.% peptide = 100-%TFA-6.45%

The total weight of the target for these experiments was calculated using the equations below. Equation 3. Total weight of the target = (13.2×10000)/(% peptide content×% purity)

The peptide was weighed into a 15 mL or 50 mL conical tube using a Mettler Toledo XP105 Delta Mass analytical balance, and the actual total weight was recorded and used to determine how much DMSO to obtain 50 mg/mL. The calculations are shown below: Equation 4. DMSO (μL) = (Actual Total Weight (mg) × 264 μL)/(Total Target Weight (mg))

The stock solution was then diluted to 2 mg/mL in an appropriate formulation buffer (1 part DMSO stock solution, 24 parts buffer).

Calculate the peptide weight and percentage content as described above and add the appropriate buffer directly to the peptide. The total weight of the target was calculated using Equation 3 and the volume of buffer used to obtain 2 mg/mL peptide was calculated using Equation 5. Equation 5. Buffer (ml) = (Actual Total Weight (mg) × 6.6 mL)/(Total Target Weight (mg))

The peptide was further diluted 1:4 with buffer to obtain 0.4 mg/mL or the buffer was directly added to the anhydrous peptide to obtain 0.4 mg/mL only when specified. In the latter case, Equation 6 is used to determine the appropriate volume to add. Equation 6. Buffer (ml) = (Actual Total Weight (mg) × 33 mL)/(Total Target Weight (mg))

The peptide was dissolved by inverting the conical tube and not by sonication or vortexing.

To pool 5 peptides, equal volumes of 2 mg/mL stock solutions were combined to obtain 0.4 mg/mL of each peptide. In the case of pools with less than 5 peptides, equal volumes of peptides were combined and the solution was diluted with an appropriate buffer to obtain 0.4 mg/mL of each peptide. Invert the assembly 3 to 5 times to mix. Transfer the formulated peptide to a glass bottle to observe solubility. Take pictures every two hours to record any changes in appearance.

Poly ICLC is commercially available. Combine 150 µL poly-ICLC and 450 µL peptide aggregates in a 2 mL glass vial at a ratio of peptide to poly-ICLC 3:1. The solution was inverted 3 to 5 times to mix, and photography was taken every two hours for 6 hours to record any changes in appearance. All pH measurements are performed using a Mettler Toledo inLab Micro pH meter, which is calibrated daily before use. The 100 µL sample analyzed was removed and added to the microcentrifuge tube to measure the pH. Then discard the sample. The sample was subjected to UPLC-MS (Waters Acquity H-Class with Acquity QDa mass spectrometer). Repeat the analysis of the 2 µL injection of each sample using an 8-minute gradient from 10:90 solvent A:B to 50:50 solvent A:B (A: 0.1% TFA/water, B: 0.1% TFA: acetonitrile). The initial solubility of the peptide in the standard formulation was determined for each peptide at 0.4 mg/mL and 2 mg/mL. Although photographs were taken, they did not always show solubility clearly because the gel was usually clear or the peptide maintained small glassy particles when it had dissolved. The peptide solubility in 5 mM SS/D5W containing 4% DMSO is indicated in Table 10.

Table 10. Peptide solubility in 5 mM SS/D5W containing 4% DMSO and observation results

Peptides insoluble in 5 mM SS were tested using 0.25 mM SS/D5W containing 4% DMSO. The results are summarized in Table 11. Many of these peptides insoluble in 5 mM SS were soluble in 0.25 mM SS/D5W containing 4% DMSO at a concentration of 0.4 mg/mL after 6 hours and this lower SS concentration test was used in further studies.

Table 11. Peptide solubility in 0.25 mM SS/D5W containing 4% DMSO and observation results

Based on these results, peptides L7, L8, L9, L14, L10c, L11d, L11f and L15 were selected for formulation studies. Testing DMSO-free formulations as a way to increase the stability of formulated peptides and slow the dimerization of cysteine-containing peptides. All peptides were tested in 0.25 mM SS (L7, L8, L9, L14, L10c, L11d, L11f and L15) at a concentration of 0.4 mg/mL and were soluble after 6 hours without DMSO. The peptides L7, L8, L9, L10c, L12d and L14 were tested in 5 mM SS and were soluble after 6 hours without DMSO. The pH values of the peptide formulations in 0.25 mM SS/D5W and 5 mM SS/D5W are listed in Table 12.

Table 12. pH of 0.4 mg/mL peptide formulation in 0.25 mM SS/D5W and 5 mM SS/D5W

Because the peptides reported in Table 11 were all soluble in 0.25 mM SS, the initial SS design was studied using lower SS concentrations. It is also considered that the aggregate is designed to have individual solubility. Because L15 and L11f are soluble in low SS, these two peptides are brought together. The initial three aggregate designs are shown in Table 13.

Table 13. Initial peptide pools in 0.25 mM SS/D5W

All peptides remained soluble after assembly. The pH values of the peptide aggregates with or without the addition of polyICLC are provided in Table 14.

Table 14. pH of peptide aggregates from Table 12 with or without polyICLC

The aggregates from each design were then mixed with poly ICLC to test their compatibility with adjuvants. When combined with poly ICLC, assembly 3 and each assembly containing peptide L10c precipitated. In addition, the pool with 3 peptides had a pH lower than 5.0 when combined with polyICLC, which indicates that the buffer capacity should be higher when the pool contains more than two peptides.

Because L7, L8, L9, L10c, and L14 are all soluble in 5 mM SS, they were tested in aggregates with higher SS concentrations. Three aggregates were tested with these five peptides. One assembly does not contain L10c, one has only L10c and the third has all five peptides. Because L11f and L15 are insoluble in this higher concentration, they are formulated in 0.25 mM SS. However, due to the observed precipitation, it was separately formulated to 0.4 mg/mL rather than in a single aggregate. Each of the peptides is soluble in its corresponding formulation. Based on visual inspection and after the pool and polyICLC are combined, the pH is all between 5.0 and 6.3 (Table 15). These pools are also compatible with polyICLC, which is suitable for subcutaneous injection.

Table 15. pH of peptide aggregates without poly ICLC vs. pH of peptide aggregates with poly ICLC

The peptide L11i was tested in D5W with various succinate concentrations in the absence of DMSO. At 0.4 mg/mL, the peptide appeared soluble in all concentrations of SS. Some precipitation was observed at higher SS concentrations at 2 mg/mL. All samples looked the same when combined with poly ICLC. Formulation of 2 mg/mL or 0.4 mg/mL peptide L11i in 0.25 mM SS, 0.5 mM SS or 5 mM SS without poly ICLC and 0.4 mg/mL peptide L11i with poly ICLC in 0.25 mM SS, 0.5 mM The pH of the formulation in SS or 5 mM SS is shown in Table 16.

Table 16. 2 mg/mL or 0.4 mg/mL L11i peptides without poly ICLC in 0.25 mM SS, 0.5 mM SS and 5 mM SS and 0.4 mg/mL peptide L11i with poly ICLC in 0.25 mM SS, 0.5 mM PH in SS and 5 mM SS

Finalized aggregates based on results include aggregate 1 (L7, L8, L9, L10c, and L14 in 5 mM SS/D5W), aggregate 2 (L11i or L11f in 0.25 mM SS/D5W), and aggregate 3 (L15 In 0.25 mM SS/D5W). Test the retention rate of each of these aggregates on a 0.2 µm filter from Pall (HP1002). The pre-filtered samples and the samples after each of the 2 filtrations were analyzed by UPLC-MS. Less than 3% of L11f and L11i are lost after the first filtration step and no additional peptide is lost after the second filtration step. Only 4.9% L15 was lost after the first filtration and then 1.3% was lost after the second filtration. Less than 3% of each peptide in aggregate 1 was lost after two filtration steps. in conclusion

The solubility of a series of potential GATA3 peptides in a formulation buffer containing 5 mM SS/D5W containing 4% DMSO was tested. Peptides insoluble in 5 mM SS were also tested at lower SS concentrations. Based on these results, seven peptides were selected, five of which were soluble in 5 mM SS (L7, L8, L9, L10c, and L14) and others were soluble in lower concentrations (L11f, L11i, and L15). The removal of DMSO is also tested and can increase solubility and slow down disulfide formation which can make UPLC analysis more difficult. Each of the selected peptides is soluble without DMSO.

Based on the solubility results, 3 aggregate designs were generated using 0.25 mM SS/D5W. Although these aggregates are soluble, some are not compatible with poly-ICLC. In some cases, precipitation was observed when the aggregate containing L10c was mixed with poly-ICLC. The same observation results were obtained when the aggregates containing L11f and L15 were combined with poly-ICLC. Based on these results, the fourth group of aggregates is designed. The first assembly contains L7, L8, L9, L10c and L14 in 5 mM SS/D5W and is compatible with poly-ICLC. The peptides L15 and L11f or L11i were kept as individual peptides to be prepared by directly dissolving them with 0.25 mM SS/D5W at 0.4 mg/mL. These aggregates are all greater than pH 5.0 when mixed with poly-ICLC, which is acceptable for subcutaneous injection.

Although the preferred embodiments have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided as examples only. Those skilled in the art will now think of many changes, changes, and substitutions without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. The following patent application scope is intended to define the scope of the present invention, and thus covers methods and structures within the scope of these patent applications and their equivalents.

The novel features of the present invention are elaborated in the appended patent application. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description of illustrative embodiments that utilize the principles of the present invention and the accompanying drawings:

Figure 1 shows that all twenty peptides soluble in a succinate concentration of 5 mM in the pharmaceutical composition remained soluble when the succinate concentration was reduced to 0.75 mM, 0.5 mM, and 0.25 mM.

Figure 2 shows that fourteen of the twenty peptides insoluble in the pharmaceutical composition having a succinate concentration of 5 mM became soluble at lower succinate concentrations. Of the fourteen peptides that became soluble at a lower succinate concentration, three were soluble only at a succinate concentration of about 0.25 mM.

Figure 3 shows that a pharmaceutical composition containing three different succinate concentrations (5 mM, 0.5 mM, and 0.25 mM) containing a combination of peptide and poly-ICLC in a 3:1 ratio produces a cloudy suspension similar to poly-ICLC alone , And no precipitation of poly-ICLC nucleic acid.

Figure 4 shows a graph plotting the HYDRO and pI values of the neoantigen peptide set shown in Figures 2 and 3 and described herein.

FIG. 5 shows a graph plotting the hydrophobicity and pI value of the peptides calculated based on the Kate-Dualit method shown in FIGS. 2 and 3 and the novel antigen peptide set described herein.

Claims (91)

A pharmaceutical composition comprising: (a) One or more neoantigenic peptides or their pharmaceutically acceptable salts; (b) pH regulators present at a concentration of less than 1.0 mM; and (c) Pharmaceutically acceptable carriers. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a vaccine composition. The pharmaceutical composition according to claim 1 or claim 2, wherein the pharmaceutical composition is aqueous. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigen peptides has pI>5 and HYDRO<-6. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigen peptides has pI>8 and HYDRO>-8. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigen peptides has pI <5 and HYDRO> -5. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigen peptides has pI>9 and HYDRO<-8. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigen peptides has a HYDRO value of pI> 7 and> -5.5. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigenic peptides has pI <4.3 and -4 ≤ HYDRO ≥ -8. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigen peptides has pI> 0 and HYDRO <-8, pI> 0 and HYDRO> -4, or pI> 4.3 and -4 ≤ HYDRO ≥ -8. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigen peptides has pI> 0 and HYDRO> -4, or pI> 4.3 and HYDRO ≤ -4. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigen peptides has pI> 0 and HYDRO> -4, or pI> 4.3 and -4 ≤ HYDRO ≥ -9. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigen peptides has 5 ≤ pI ≥ 12 and -4 ≤ HYDRO ≥ -9. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigen peptides has 5 ≤ pI ≥ 12 and -6 ≤ HYDRO ≥ -9. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigenic peptides has 7.5 ≤ pI ≥ 12 and -4 ≤ HYDRO ≥-9. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigenic peptides has a pI of 5 to 6.5 or 7.5 to 12 and 4 ≤HYDRO ≥ -9. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigenic peptides has 5 ≤ pI ≥ 12 and has HYDRO of -4 to -4.9 or -5.8 to -9. The pharmaceutical composition of claim 1 or 2, wherein at least one of the one or more neoantigenic peptides has a pi of 5 to 6.5 or 7.5 to 12 and a HYDRO of -4 to -4.9 or -5.8 to -9. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigen peptides is insoluble in the corresponding pharmaceutical composition, and the corresponding pharmaceutical composition contains a pH adjusting agent at a concentration of 1.0 mM or higher. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition comprises at least two neoantigen peptides. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition comprises at least three neoantigen peptides. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition comprises at least four neoantigen peptides. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition comprises at least five new antigen peptides. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition comprises at most 40 new antigen peptides. The pharmaceutical composition according to claim 1 or 2, wherein each of the one or more neoantigen peptides is soluble in the pharmaceutical composition. The pharmaceutical composition according to claim 1 or 2, wherein each of the one or more neoantigenic peptides contains 6 to 50 amino acids, 6 to 45 amino acids, 6 to 40 amino acids, 6 to 35 amino acids, 6 to 30 amino acids, 6 to 25 amino acids, 6 to 20 amino acids, 8 to 50 amino acids, 8 to 45 amino acids, 8 to 40 amino acids Amino acids, 8 to 35 amino acids, 8 to 30 amino acids, 8 to 25 amino acids, 8 to 20 amino acids, 14 to 50 amino acids, 14 to 45 amino acids Acid, 14 to 40 amino acids, 14 to 35 amino acids, 14 to 30 amino acids, 14 to 27 amino acids, 14 to 25 amino acids, or 14 to 20 amino acids. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigenic peptides contains 14 to 30 amino acids. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigenic peptides has a length of less than 15 amino acids or less, a length of 7 to 11 amino acids, or a length of 8 to 10 amino acids. The pharmaceutical composition according to claim 1 or 2, wherein at least one of the one or more neoantigenic peptides has a length of 40 amino acids or less, a length of 6 to 25 amino acids, and a length of 14 to 30 Amino acids, or 14 to 27 amino acids in length. The pharmaceutical composition according to claim 1 or 2, wherein the pH adjusting agent is a base. The pharmaceutical composition according to claim 1 or 2, wherein the pH adjusting agent is a weak acid conjugate base. The pharmaceutical composition according to claim 1 or 2, wherein the pH adjusting agent is a pharmaceutically acceptable salt. The pharmaceutical composition according to claim 1 or 2, wherein the pH adjusting agent is a dicarboxylic acid salt or a tricarboxylic acid salt. The pharmaceutical composition according to claim 1 or 2, wherein the pH adjusting agent is citric acid and/or citrate. The pharmaceutical composition according to claim 34, wherein the citrate is disodium citrate and/or trisodium citrate. The pharmaceutical composition according to claim 1 or 2, wherein the pH adjusting agent is succinic acid and/or succinate. The pharmaceutical composition according to claim 36, wherein the succinate salt is disodium succinate and/or monosodium succinate. The pharmaceutical composition according to claim 37, wherein the succinate salt is disodium succinate hexahydrate. The pharmaceutical composition according to claim 1 or 2, wherein the pH adjusting agent is present at a concentration of 0.75 mM or lower. The pharmaceutical composition according to claim 1 or 2, wherein the pH adjusting agent is present at a concentration of 0.50 mM or less. The pharmaceutical composition according to claim 1 or 2, wherein the pH adjusting agent is present at a concentration of 0.25 mM or less. The pharmaceutical composition according to claim 1 or 2, wherein the pH adjusting agent is present at a concentration of 0.25 mM to 1.0 mM. The pharmaceutical composition according to claim 1 or 2, wherein the pH adjusting agent is present at a concentration of 0.50 mM to 1.0 mM. The pharmaceutical composition according to claim 1 or 2, wherein the pH adjusting agent is present at a concentration of 0.75 mM to 1.0 mM. The pharmaceutical composition according to claim 1 or 2, wherein the pH adjusting agent is present at a concentration of 0.50 mM to 0.75 mM. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutically acceptable carrier contains a liquid. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutically acceptable carrier comprises water. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutically acceptable carrier comprises sugar. The pharmaceutical composition according to claim 48, wherein the sugar comprises dextrose. The pharmaceutical composition of claim 49, wherein the dextrose is present at a concentration of 5% w/v. The pharmaceutical composition of claim 48, wherein the sugar comprises trehalose. The pharmaceutical composition according to claim 48, wherein the sugar comprises sucrose. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutically acceptable carrier comprises dimethyl sulfoxide (DMSO). The pharmaceutical composition of claim 53, wherein the DMSO is present at a concentration of 0.1% to 10%, 0.5% to 5%, or 1% to 3%. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is lyophilizable. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition further comprises an immunomodulator or adjuvant. The pharmaceutical composition according to claim 56, wherein the immunomodulator or adjuvant is selected from the group consisting of: 1018 ISS, aluminum salt, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, ARNAX, STING agonist Agent, dSLIM, GM-CSF, IC30, IC31, imiquimod (Imiquimod), ImuFact IMP321, IS patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, carrier system, PLGA particles, resiquimod, SRL172, virus particles And other virus-like particles, YF-17D, VEGF capture agent, R848, β-glucan, Pam3Cys and Aquila QS21 stimulator. A pharmaceutical composition comprising: (a) One or more neoantigenic peptides; (b) Dimethyl sulfonate present in a concentration of at least 0.5%, 1%, 2%, 3% or higher; (c) sugar present in a concentration of at least 1% w/v, 2% w/v, 3% w/v, 4% w/v, 5% w/v or higher; and (d) Succinic acid or succinate present at a concentration of less than 1.0 mM. A pharmaceutical composition comprising: (a) One or more neoantigenic peptides; (b) 1% to 10% dimethyl sulfoxide; (c) 1% w/v to 10% w/v sugar solution; and (d) Less than 1.0 mM succinic acid or succinate. A pharmaceutical composition comprising: (a) One or more neoantigenic peptides; (b) 1% to 10% dimethyl sulfoxide; (c) 1% w/v to 10% w/v sugar solution; and (d) less than 1.0 mM succinic acid or succinate; and (e) Poly I: Poly C. 2 and The pharmaceutical composition of any one of 58 to 60, wherein the pharmaceutical composition further comprises sodium chloride. A pharmaceutical composition comprising: (a) One to five new antigen peptides, (b) 1% to 4% dimethyl sulfoxide, (c) 3% w/v to 5% w/v dextrose, (d) Succinic acid or succinate present at a concentration of less than 1.0 mM. 2. The pharmaceutical composition of any one of 58 to 60 and 62, wherein each of the one or more neoantigenic peptides is at least 1 μg/mL, at least 10 μg/mL, at least 25 μg/mL, at least 50 μg /mL or at least 100 μg/mL. 2. The pharmaceutical composition of any one of 58 to 60 and 62, wherein each of the one or more neoantigen peptides is at most 5000 μg/mL, at most 2500 μg/mL, at most 1000 μg/mL, at most 750 μg /mL, at most 500 μg/mL, at most 400 μg/mL, or at most 300 μg/mL. 2. The pharmaceutical composition of any one of 58 to 60 and 62, wherein each of the one or more neoantigen peptides ranges from 10 μg/mL to 5000 μg/mL, 10 μg/mL to 4000 μg/mL, 10 Concentrations from μg/mL to 3000 μg/mL, 10 μg/mL to 2000 μg/mL, 10 μg/mL to 1000 μg/mL, 25 μg/mL to 500 μg/mL or 50 μg/mL to 300 μg/mL exist. 2. The pharmaceutical composition of any one of 58 to 60 and 62, wherein a higher percentage of the neoantigen peptide is soluble in the pharmaceutical than the pharmaceutical composition containing a pH adjusting agent at a concentration of 1.0 mM or higher combination. 2. The pharmaceutical composition of any one of 58 to 60 and 62, wherein there is a higher percentage of the one or more new antigens as compared to the pharmaceutical composition containing the pH adjusting agent in a concentration of 1.0 mM or higher The peptide is soluble in the pharmaceutical composition. A vaccine comprising the pharmaceutical composition according to any one of claims 1 to 67. If the vaccine of claim 68, it is a neoplastic vaccine. An immunogenic composition comprising the pharmaceutical composition according to any one of claims 1 to 67. A vaccination or immunization kit includes: (a) A lyophilized composition comprising one or more neoantigenic peptides as in any one of claims 1 to 67; (b) pH adjusters; and (c) Instructions for combining (a) and (b) into a solution, where the solution contains the pH adjusting agent at a concentration of less than 1.0 mM. A vaccination or immunization kit includes: (a) A composition comprising one or more lyophilized neoantigen peptides or pharmaceutically acceptable salts thereof; (b) pH adjusters; and (c) Instructions for combining (a) and (b) into a solution, where the solution contains the pH adjuster at a concentration of less than 1.0 mM. The vaccination or immunization kit of claim 71, wherein the kit further contains an adjuvant. The vaccination or immunization kit of any one of claims 71 to 73, wherein the kit further contains a pharmaceutically acceptable carrier. For the vaccination or immunization kit of claim 74, wherein the pharmaceutically acceptable carrier is water. A method for preparing a pharmaceutical composition according to any one of claims 1 to 67, which comprises combining one or more new antigen peptides with a pH adjusting agent and a pharmaceutically acceptable carrier to form a peptide solution, wherein the pH is adjusted The agent is present at a concentration below 1.0 mM. A method for preparing a pharmaceutical composition, comprising combining one or more lyophilized neoantigen peptides with a pH adjusting agent and a pharmaceutically acceptable carrier to form a peptide solution, wherein the pH adjusting agent is at a concentration of less than 1.0 mM exist. The method of claim 76 or 77, wherein the method further comprises filtering the peptide solution. The method of claim 76, wherein the one or more neoantigenic peptides are lyophilized before merging. A method for preparing a vaccine, which comprises combining the pharmaceutical composition according to any one of claims 1 to 67 with an immunomodulator or adjuvant. Use of a pharmaceutical composition according to any one of claims 1 to 67 for the manufacture of a medicament for the treatment of a condition or disease. Use according to claim 81, wherein the medicament further comprises or is used in combination with another therapeutic agent. The use according to claim 81 or 82, wherein the medicament further comprises or is used in combination with a second pharmaceutical composition, wherein the second pharmaceutical composition is the pharmaceutical combination according to any one of claims 1 to 67 Thing. The use according to claim 83, wherein the medicament further comprises or is used in combination with a third pharmaceutical composition, wherein the third pharmaceutical composition is the pharmaceutical composition according to any one of claims 1 to 67. The use according to claim 84, wherein the medicament further comprises or is used in combination with the fourth pharmaceutical composition, wherein the fourth pharmaceutical composition is the pharmaceutical composition according to any one of claims 1 to 67. The use according to claim 85, wherein the medicament further comprises or is used in combination with at least a fifth, sixth, seventh or eighth pharmaceutical composition, wherein the at least The fifth, sixth, seventh or eighth pharmaceutical composition is the pharmaceutical composition according to any one of claims 1 to 67. For use according to claim 81 or 82, wherein the condition or disease is a neoplasm. The use according to claim 87, wherein the neoplasm is cancer. A new antigen peptide aqueous solution, which contains: (a) one or more neoantigenic peptides; and (b) Succinic acid or succinate present at a concentration of less than 1.0 mM. A method for preparing a new antigen peptide aqueous solution, which comprises combining one or more new antigen peptides with succinic acid or succinate and a liquid carrier, wherein the succinic acid or succinate is present at a concentration of less than 1.0 mM . A method for increasing the solubility of one or more neoantigen peptides, which comprises adjusting the concentration of a pH adjusting agent present in a pharmaceutical composition, the pharmaceutical composition comprising one or more neoantigen peptides, the pH adjusting agent and a pharmaceutically acceptable Carrier; wherein the adjustment includes reducing the concentration of the pH adjusting agent to a concentration lower than 1.0 mM.

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